Hydroxyproline is found in high concentrations in connective tissue proteins. It is remarkably useful to detect it because variation in urine levels of hydroxyproline is associated with various diseases. High performance liquid chromatography methods, in which ultraviolet and electrochemical detectors were used, were developed and validated for the determination of hydroxyproline in urine. Both methods included acid hydrolysis and derivation. The most appropriate time, temperature and pH were identified to optimize derivatization processes.