The present study was focused on determining the effect of L-tryptophan on quality of sperm and its fertilizing capacity in endangered trout Salmo coruhensis, Anatolian trout Salmo rizeensis and rainbow trout Oncorhynchus mykiss. Different activation media (NaCl, 0.3%; NaHCO3, 1%) were supplemented with L-tryptophan [Control (0), 0.5, 1, 2, 3, 4 and 5 mM] for assessing sperm motility and its duration, fertility and hatching rate of eggs. The results from the present study indicated that addition of L-tryptophan increased the motility rate and duration in O. mykiss, S. rizeensis and S. coruhensis compared to control group. Highest sperm motility rate and its duration, and fertility and hatching rate of eggs of O. mykiss, S. rizeensis and S. coruhensis were 0.5 mM, 5 mM and 2 mM, respectively. To conclude, sperm quality and fertility were positively affected by quantitative changes different concentrations of L-tryptophan. In addition, L-tryptophan can be used in activation medium for O. mykiss, S. rizeensis and S. coruhensis.