Although banned, dyes, such as Victoria pure blue BO (VPBO), are illicitly used in aquaculture to treat or prevent infections due to their therapeutic activities. The present study examined the formation of phase I and phase II metabolites derived from VPBO using trout liver microsomes and S9 proteins. The well-known malachite green (MG) dye was also studied as a positive control and to compare its metabolism with that of VPBO. First, we optimised the incubation conditions for the detection of VPBO and MG metabolites by studying the formation of cytochrome P450 (CYP) substrates. Using the determined conditions (2 h at 20 °C), we incubated VPBO with trout microsomal and S9 fractions induced with β-naphtoflavone, and analysed the supernatant in a LC-LTQ-Orbitrap-HRMS system. The in vitro assays led to the detection of 16 VPBO metabolites from Phase I reactions, arising in particular from reactions with CYP1A. No metabolites were detected from Phase II reactions. The main metabolite detected, deethyl-VPBO, was CID-fragmented to determine its chemical structure, and thus recommend a potential biomarker for the control of VPBO in farmed fish foodstuffs.