Abstract A high-performance liquid chromatographic method was developed to assay penicillin G (pen-G) in chinook salmon muscle tissue. Solid-phase extraction techniques were used to recover pen-G and the internal standard, phenoxymethylpenicillin, from the salmon tissue samples. Pen-G was analyzed using a 25 cm × 4.6 mm I.D. Ultrasphere OS (C 8) column and a mobile phase of acetonitrile—0.02 M phosphate buffer, pH 6.75 (43:200) with ultraviolet detection at 214 nm. A linear calibration curve ( r 2 = 0.9994) was obtained for pen-G over the range 0.05–3.0 ppm. Recoveries of pen-G added to tissues at 0.1, 1.0 and 2.0 ppm were 63.9%, 64.2% and 65.4%, respectively, in salmon muscle tissue, and intra-assay variabilities of 4.89% at 1.0 ppm and 2.79% at 2.0 ppm were optimized for salmon muscle tissue. The detection limit of pen-G was 0.05 ppm in salmon muscle tissue, using a signal-to-noise ratio of 5:1.