A modified assay for the determination of hydroxyproline in tissue is presented. The modifications greatly reduce the time required for analysis of excised tissue as first introduced by Stegemann and Stalder [1]. These modifications include a change in the technique for tissue hydrolysis and a change in the preparation of the hydroxyproline oxidizing agent. The analysis utilizes the standard addition technique, eliminating the need for correction of matrix effects between the specimen and standard. This paper attempts to give a complete detailed description of the assay such that the procedure may be repeated without requiring additional reference material.