Florfenicol is commonly used to treat bacterial diseases in striped catfish Pangasianodon hypophthalmus aquaculture with limited knowledge of pharmacokinetic database, explaining treatment failures experienced by farmers. Striped catfish (100 g) were fed medicated feed (10 mg florfenicol/kg fish) by oral gavage. Plasma, liver, and kidney were collected up to 96.0 h after drug administration and analyzed for florfenicol. A single oral gavage dose of 10 mg florfenicol/kg fish revealed a rapid elimination, demonstrated by an elimination half-life (T1/2el) of 2.56 h. Florfenicol amine was found in liver and kidney tissue. The calculation of T > MIC (time that the concentration remains above the minimum inhibitory concentration) revealed a potential therapeutic effect on bacillary necrosis, a common disease in striped catfish if the dosing interval is 12 h. The depletion of florfenicol in striped catfish muscle was determined in feeding trials with medicated feed containing florfenicol at 10 mg/kg fish for five consecutive days in tank and pond conditions. The fast elimination of florfenicol resulted in a withdrawal time of two days (or 39 degree days) in proposed tank-rearing conditions and four days (or 112 degree days) in pond-rearing conditions.