To establish a method based on alkaline hydrolysis for determination of L-hydroxyproline(L-Hyp) in hydrolyzed animal protein, in which NaOH solution and polytetrafluoroethylene(PTEF) hydrolysis tube with cover were used. Method The sample was hydrolysed by 6 ml 2.5 mol/L NaOH soultion in 10 ml hydrolysis tube and heated in the oven at 110 ℃ for 2 h. The hydrolysate of L-Hyp was oxidized by chloramine-T and then reacted with paradime thylaminobenzaldehyde to generate red complex. The absorbency was determined at (558±2) nm. ResultsUnder the optimal conditions, the calibration curve was linear in the range of 0-10 μg/ml with detection limit of 1.35 μg/g, and the relative standard deviation(RSD) was 1.0%-2.3%. It was effective in the determination of L-Hyp in milk and dairy products with recoveries of 88.7%-96%. Conclusion Compared with acidolysis, the alkaline hydrolysis had more simple experimental procedure, fewer measurement time, highly sensitive and reproducible with consistency.