The monastery is located in the center of the ancient settlement of Pereyaslav, north of the citadel. From 1695 to 1700, the stone Cathedral of the Ascension of the Lord was built here, financed by Hetman Ivan Mazepa. Wooden structures were erected around the cathedral, forming the architectural ensemble of the new male monastery. In 1700, after the restoration of the episcopal see that existed during the era of Kievan Rus, the Ascension Monastery became the residence of the ruling archbishop, contributing to the formation of a new city center. Until 1733 and from 1785, the Pereyaslav bishop served as a vicar to the Metropolitan of Kyiv, and in the interval between these dates, he was the archbishop of an independent diocese that included not only Pereyaslav but also the Orthodox population of Right-Bank Ukraine, which remained under the rule of Catholic Poland until the end of the 18th century. From 1785 to 1793, the center of the diocese was temporarily in the city of Slutsk, in 1799 it was renamed to the Little Russian-Pereyaslav Diocese, in 1803 to the Poltava-Pereyaslav Diocese, and in 1847 the see was moved to Poltava. Until the early 20th century, Pereyaslav was part of Poltava region. In 1816, the monastery's brotherhood was transferred to Saratov, after which the Ascension Monastery was considered non-staff, and in 1847 it was attached to the Poltava Archiepiscopal House.

In 1701, Mazepa gifted the monastery the Peresopnytsia Gospel, before which the Presidents of Ukraine take an oath. This manuscript book, known for its artistic design, contains the first known translation of the canonical Gospel into the Ukrainian language. The Gospel was created in Volhynia: part of the manuscript was completed in 1556 at the women's Palace Monastery near Iziaslav, and another part in 1561 at the men's Peresopnytsia Monastery in Rivne region. The work on the book was funded by Princess Anastasia Zaslavska-Olshanska, who took monastic vows under the name of Paraskeva. In the 1830s, the book was discovered in the library of the Pereyaslav Seminary by philologist Joseph Bodiansky, whose publications initiated the scientific study of the Peresopnytsia Gospel as an outstanding monument of spiritual literature in Ukraine. After the seminary was moved to Poltava, the book left Pereyaslav and was subsequently kept in the Poltava Historical and Local Lore Museum, and after the war, in the Kyiv-Pechersk Lavra Museum-Reserve, from where it was transferred to the National Library of Ukraine named after Volodymyr Vernadsky.

Since 1738, a Collegium operated at the monastery, one of the highest schools in Ukraine in the 18th century, opened by Pereyaslav Bishop Arseniy Berlo. Education at the Collegium was available to all classes, starting at the age of 11 and lasting 11-12 years. About half of the students came from low-income families and lived in the bursary funded by the diocese. In the lower classes, Church Slavonic, Latin, and Greek languages, catechism, basics of Christian faith, arithmetic, and geography were studied. In the middle classes, poetics and rhetoric were taught, and in the higher classes, philosophy and theology. In 1753-1754, Ukrainian philosopher Hryhoriy Skovoroda taught poetics at the Collegium. In 1776, the Collegium was transformed into a spiritual seminary, which was moved to Poltava in 1862. Initially, the Collegium was located in a wooden house that burned down in 1748, after which classes were moved to an empty private house. In 1753-1757, a stone building of the Collegium was constructed, which still exists today. From 1862, a spiritual school was located here, and in 1972, the Memorial Museum of Hryhoriy Skovoroda was opened.

On October 4, 1766, the head of Saint Martyr Daniel Kushnir, the elder of the Assumption Church of the locality of Mliiv, who was executed for returning the church to Orthodoxy, was buried in the Ascension Cathedral. His memory began to be honored immediately after his death, and in 1994, the Holy Synod of the UOC canonized him, establishing a feast day on August 11. Since 1786, the relics of the Venerable Martyr Makariy Ovruchsky and Kanivsky, who was killed by the Turks in 1678, were located in the cathedral. In 1942, the relics were transferred to Cherkasy, and the memory of Makariy is celebrated on September 7 and May 13.

Since the early 1920s, the Ascension Cathedral belonged to the UAPC, in 1925 the territory of the monastery was occupied by a military unit, and in the early 1930s, the church was turned into a grain storage facility. During the war, the cathedral lost its dome, and cracks appeared in the walls. In 1946, the monastery buildings were recognized as architectural monuments of national significance. From 1954 to 1964, the cathedral was restored, but the frescoes were whitewashed, and it was used as a warehouse. A water tower was set up in the bell tower. In 1955, a protective zone for the monument was established, but part of the territory remained with the military. In 1972, the Memorial Museum of Hryhoriy Skovoroda was opened in the Collegium building, and in 1975, a Diorama Museum dedicated to the crossing of the Dnieper by Soviet troops in 1943 was opened in the cathedral. Since 1978, construction of a new Historical Museum was underway, but the project was not completed due to lack of funding. In 2002, the process of transferring the territory of the military unit to the National Historical and Ethnographic Reserve 'Pereyaslav' began. In the late 1990s, a community of the UOC-KP was registered at the cathedral, whose members gathered in a specially equipped room, later in the bell tower. To ensure the revival of the shrine occurred within the framework of the canonical Church, on February 10, 2011, the Holy Synod of the UOC blessed the opening of the Ascension Male Monastery.