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Using a commercially available testosterone enzyme immunoassay (EIA), we developed and validated an assay procedure for determining testosterone levels in small-volume (20 μL) avian plasma samples. We evaluated this EIA’s utility by measuring plasma testosterone levels in Mourning Doves (Zenaida macroura), White-eyed Vireos (Vireo griseus), Red-eyed Vireos (Vireo olivaceus), and Indigo Buntings (Passerina cyanea). Standard biochemical validations (e.g., parallelism, recovery of exogenous testosterone) demonstrated that the assay accurately and precisely measured testosterone in avian plasma. We compared plasma testosterone levels in males and females of all four species and Indigo Buntings in various reproductive stages to physiologically validate the assay’s ability to determine biologically important changes in testosterone levels. Plasma testosterone levels were higher in males compared to females in three of four species. Prebreeding and breeding male Indigo Buntings had higher circulating testosterone levels than postbreeding males. Testosterone levels in our study were similar to reported values for other passerine species using radioimmunoassay procedures. Our results suggest that this EIA procedure is very effective for determining testosterone levels in small-volume avian plasma samples and is sensitive enough to detect biologically important changes in the gonadal activity of birds. Thus, this assay has considerable utility for measuring testosterone in small birds (<15 g), from which only small volumes of plasma (20 μL) can be collected.