Endocrine studies on small mammals typically use blood draws for plasma analysis. However, in larger mammals urinary steroids can be used to track an individual’s reproductive state. Urinary steroid analysis can also be ideal for smaller animals as it is non-invasive and allows for repeated testing. Big brown bats (Eptesicus fuscus) are small insectivores that have an annual reproductive cycle. As such, repeated blood draws are not ideal for the species; however, the use of a captive breeding colony provides the opportunity for repeated urine collection. The present study aimed to quantify endogenous levels of unconjugated 17beta-estradiol (E₂) via urinary analysis in male and female big brown bats. Urine was quantified as unadjusted steroid measurements as well as creatinine-corrected values to control for animal hydration and activity levels. We observed differences in urinary E₂ between male and female adults, as well as age effects in males. A number of seasonal differences in both creatinine-adjusted and unadjusted urinary E₂ were observed in males and non-pregnant females. We also explored seasonal differences in urinary progesterone (P₄) levels in non-pregnant female bats across seasons. The highest values of urinary steroids were measured in pregnant female bats were both E₂ and P₄ peaking ~20 days prior to parturition. This project is the first to monitor urinary steroids across the reproductive cycle in bats, and is the first step towards using urinary steroids to detect pregnancy in bats. Importantly, the quantification of urinary steroid levels is critical in interpreting their role as possible reproductive pheromones in bats.