The Siberian flying squirrel (Pteromys volans) spends almost all of its life on trees and thus depends extremely highly on forests. Forest fragmentation has been reducing its habitat. To conserve the squirrel’s populations in fragmented landscapes we need information on not only its ecology but also its physiology, because stress and poor body condition resulting from clear-cutting could negatively influence its reproductive physiology. However, there is a paucity of information on the Siberian flying squirrel’s basic reproductive physiology, and there is no established method for studying it. Our goal was to validate fecal progesterone analysis in this animal using a commercial enzyme immunoassay (EIA) kit for endocrine profiles in Siberian flying squirrels. First, we tested parallelism between serially diluted fecal progesterone and a standard curve to validate the EIA. Comparison of the slopes of the two regression lines to test for parallelism revealed no significant difference. Therefore, progesterone concentrations in the fecal samples of the Siberian flying squirrels were exactly measured. Second, we compared progesterone concentrations among four groups—pregnant females, adult females in the non-breeding season, juvenile females, and adult males—to determine whether fecal progesterone analysis was useful for evaluating reproductive status using GLMM. Fecal progesterone concentrations were significantly higher in pregnant females than in other groups. These results indicated that fecal progesterone analysis in Siberian flying squirrels was valid for predicting pregnancy.