A high performance liquid chromatographic (HPLC) method was developed for the determination of trenbolone acetate, 17α-trenbolone and 17β-trenbolone in bovine muscle and liver. Bovine muscle sample was extracted with acetonitrile, filtered, and defatted with acetonitrile-saturated n-hexane. The acetonitrile extract after concentration and clean-up with Bond Elut C18 cartridge was ready for HPLC analysis. HPLC conditions were as follows, column: Inertsil ODS 3V, mobile phase: acetonitrile/methanol/H2O (50:10:40, v/v), flow rate: 1 mL/min, and detecting wavelength: UV 340 nm. For bovine muscle, the recoveries were 83.8~98.9% for trenbolone acetate, 17α-trenbolone and 17β-trenbolone spiked at concentrations between 2~4 ppb, and the variation coefficients were 1.2~4.8%. For bovine liver, the recoveries were 82.6~95.7% for the three trenbolones spiked at concentrations between 10~20 ppb, and the variation coefficients were 1.9~6.7%. The detection limits for trenbolone acetate, 17α-trenbolone and 17β-trenbolone were 1, 0.5 and 0.5 ppb in bovine muscle, and 4, 2 and 2 ppb in bovine liver, respectively. After the survey, 30 marketed bovine muscle samples showed no detection of trenbolone acetate and its metabolites.