Non-invasive techniques were used to investigate the reproductive biology of captive squirrel gliders (Petaurus norfolcensis) for 3 months during the breeding season. The squirrel glider is a medium-sized marsupial glider of eastern Australia and is currently listed as a threatened species as a result of habitat destruction and fragmentation. Urinary cytology was used to determine the timing of oestrus, and the presence of sperm confirmed mating. Progesterone and oestradiol-17β were identified in faecal samples via thin-layer chromatography, and were used to characterise the reproductive cycle. Reproductive activity was observed in three of four females, with births occurring during June and July. A preoestrus increase in faecal oestradiol-17β was detected in a single female, whilst significant increases occurred post partum (±2 days) in two of four females, suggesting that the squirrel glider may undergo a postpartum oestrus. Faecal progesterone profiles showed low concentrations before oestrus and significantly elevated concentrations after oestrus, which were maintained throughout pregnancy. Parturition coincided with a decrease in progesterone concentrations (±1 day). This study successfully used non-invasive monitoring of urinary cytology and faecal steroids to define luteal and gestational length as 16–17 days, a previously unpublished detail.