The determination of prothrombin time (PT) has been standardized for domestic chickens; however, validated tests of coagulation are lacking for nondomestic avian species, limiting the investigation of acquired hemostatic disorders in these species. The purpose of this study was to validate the PT assay for chickens in our laboratory using both fresh and frozen plasma and to apply the assay to psittacine bird plasma to establish reference intervals for PT of Hispaniolan parrots (Amazona ventralis) and umbrella cockatoos (Cacatua alba). We used avian tissue thromboplastin, prepared from brain tissue of 2-week-old chickens, for determining all PT values. The reference intervals for PT of chickens (n = 6) were 7.5–10.6 and 7.0–11.1 seconds for fresh and frozen plasma, respectively. The reference intervals for PT of Hispaniolan parrots (n = 6) were 7.5–13.4 and 9.0–11.3 seconds for fresh and frozen plasma, respectively. The reference intervals for PT of cockatoos (n = 14) differed significantly (P < .0001) when 2 different aliquots of avian thromboplastin were used for frozen plasma (11.2–15.8 versus 10.0–13.0 seconds). These PT values should be viewed as estimates for these species because of the small sample sizes in our study. PT values for plasma samples were similar under either fresh or frozen conditions. When the same aliquot of thromboplastin was used, interspecies differences in PT were evident. Variation between aliquots of thromboplastin caused the most significant difference in PT values between avian plasma samples; thus, aliquots of avian tissue thromboplastin should be prepared in volume sufficient to insure that multiple PT assays can be performed for a single patient.