An inexpensive, harmless, and quick technique of sample preparation followed by a reversed-phase high-performance liquid chromatography (HPLC) for the simultaneous quantification of trenbolone acetate and 17beta-trenbolone in cattle muscle is presented. Analysis by HPLC with photo-diode array detector was performed using a short C1 column with an isocratic mobile phase. The method was validated by the analyses of spiked samples, resulting recoveries (> or = 87.9%; relative standard deviations < or = 3.4%), analytical total time (< 20 min/sample, where, a batch of 12 samples in 2 h), and quantitation limits (< or = 1.8 ng/g). No harmful organic solvents and reagents were used at all.