A method for the determination of malachite green and its major metabolite leucomalachite green in rainbow trout muscle is reported with limits of detection of 0.8 and 0.6 μg kg−1, respectively. Residues were extracted with an acetonitrile–acetate buffer mixture and partitioned into methylene chloride. Clean-up of the extracts was performed on alumina and propylsulfonic acid solid-phase extraction columns using the automated solid-phase extraction system. The chromatographic separation of malachite green and leucomalachite green was achieved on a Chromspher 5B column using an acetonitrile–acetate buffer mobile phase. Leucomalachite green was converted to malachite green by post-column oxidation before spectrophotometric detection at 600 nm. The mean recoveries of malachite green and leucomalachite green from control rainbow trout muscle spiked at 2–50 μg kg−1 were 65% (range 63.4–65.9%, relative standard deviation 3.9–16.1%) and 74% (range 58.3–82.6%, relative standard deviation 3.3–11.4%), respectively. Qualitative confirmation of the determined residues was performed with liquid chromatography coupled with tandem mass spectrometry detection with limits of detection of 2.5 and 1 μg kg−1 for malachite green and leucomalachite green, respectively.