A rapid and sensitive ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method was developed for the determination of cyadox (CYX) and its three major metabolites, quinoxaline-2-carboxylic acid (QCA), 1-desoxycyadox (1-DCYX) and 1,4-bisdesoxycyadox (BDCYX) in fish. Samples were extracted with acetonitrile–0.5 M hydrochloric acid (9 + 1) and cleaned up using Oasis MAX cartridges. The total time taken for separation by UPLC was less than 5 min. The four target compounds were then determined by ESI-MS/MS. At the fortified levels of 2–50 μg kg−1 in fish, recoveries of all the compounds ranged from 80.2% to 88.2%, with a relative standard deviation of 6.8 to 14.6%. Limits of detection for CYX, QCA, 1–DCYX and BDCYX were 0.52, 1.07, 0.69 and 0.38 μg kg−1, respectively.