A spectrophotometric procedure for estimation of L-lysine-alpha-oxidase activity involved measurement of optical density in samples after reaction of the H2O2 formed with o-phenylene diamine. Suitable values of pH, o-phenylene diamine concentration and time of incubation were chosen. The procedure developed was distinctly dissimilar as compared with the methods currently widely used. Advantages of the new method are due to its sensitivity, reproducibility, accessibility, relative safety and short time of the reaction performance. The procedure may be used for estimation of L-lysine content in various biological materials.