In our previous work we identified the presence of meso-zeaxanthin [(3R,3′S)-zeaxanthin] in trout flesh and skin (Nolan et al., 2014), but were not able to quantify this carotenoid with the method used at that time. In the present study, we developed a protocol that allows for the quantification of lutein and the three stereoisomers of zeaxanthin [(3R,3′R)-zeaxanthin, meso-zeaxanthin and (3S,3′S)-zeaxanthin] in fish flesh. We tested this protocol in two species of farmed trout (Oncorhynchus mykiss and Salmo Trutta), and we detected and quantified these carotenoids. The concentrations of each carotenoid detected (ranging from 1.18±0.68ngg−1 flesh for meso-zeaxanthin to 38.72±15.87ngg−1 flesh for lutein) were highly comparable for the two fish species tested. In conclusion, we report, for the first time, the concentrations of zeaxanthin stereoisomers (including meso-zeaxanthin) and lutein in trout flesh. This work adds further to the knowledge on the presence of these carotenoids in the human food chain.