A single 30 mg/kg dose of trimethoprim-sulfamethoxazole (TMS) was administered orally (n = 17) and intravenously (n = 13) in a crossover study design, with a 10 day washout period, to determine drug pharmacokinetics in healthy adult green iguanas (Iguana iguana). Blood samples were collected at 0 (pretreatment), 1, 2, 4, 8, 12, 24, 36, 48, 72, and 96 h after PO and IV drug administration, including a 6 h sample for the IV protocol. Plasma drug concentrations were determined by liquid chromatography–tandem mass spectrometry, and oral bioavailability of trimethoprim (>100%) and sulfamethoxazole (69.4%) were then established. Pharmacokinetic parameters were estimated using a two-stage, noncompartmental analysis of naïve-averaged data. Following the crossover study, a multidose drug trial was performed to determine steady-state nädir plasma drug concentrations. Green iguanas (n=10) were given 30 mg/kg TMS PO once daily for 7 days, with blood collected from each animal on the eighth day, 24 h after the final dose of TMS. Based on once daily oral dosing for 7 days, steady-state nädir drug concentrations of trimethoprim and sulfamethoxazole were 396 ± 116 and 5,290 ± 5,130 µg/l, which are below the minimum inhibitory concentrations breakpoints of TMS (≤2 µg per ml/38 µg per ml for susceptible organisms and ≥4 µg per ml/76 µg per ml for resistant organisms) for human isolates, per the Clinical and Laboratory Standards Institute. However, the ratio of trimethoprim:sulfamethoxazole remained greater than 1:40 up to 12 h after single oral dose exposure and at the 24 h sampling after multiple dosing at steady-state.