A cation-exchange high-performance liquid chromatographic method for the sensitive determination of dityrosine in hydrochloric acid-hydrolysate of insoluble protein is described. The method is based on the clean-up of the acid hydrolysate by Dowex 1×8 column chromatography and the separation of dityrosine in the eluate from the column by high-performance liquid chromatography with fluorescence detection on Hitachi Gell 3011-S. The limit of detection for dityrosine is 0.6 pmol per 10μl (the injecton volume) of the sample solution for the chromatography. This method permited the determination of dityrosine in collagen from cow skins and tendons, and should be useful for the determination of dityrosine in various tissues in biological samples.