We studied androgen production during development in nestling Eastern Screech-Owls (Megascops asio) and hypothesized that gender and hatch order might influence serum levels of testosterone and androstenedione. Testosterone levels were highest immediately after hatching and declined significantly in the 4 weeks leading to fledging. The average level of testosterone for 1–7 day-old owls was 3.99 ± 0.68 ng/ml. At 22–28 days of age, the average testosterone level for nestling owls was 0.83 ± 0.18 ng/ml. Testosterone levels did not differ between males or females. The average testosterone level for male nestlings was 2.23 ± 0.29 ng/ml and 2.39 ± 0.56 ng/ml for female nestlings. The average level of androstenedione for nestling owls was 1.92 ± 0.11 ng/ml and levels remained constant throughout development. Levels were significantly higher in males than females. The average androstenedione level was 1.77 ± 0.16 ng/ml for male nestlings and 1.05 ± 0.24 ng/ml for female nestlings. Hatching order did not affect levels of either androgen. Our results provide a foundation for future studies of androgen production by nestling owls.