Abstract A colorimetric assay of hydroxyproline in urine, based on the method of Firschein and Schill (1), has been developed. The following improvements have been achieved: 1. (i) Microscale techniques permitting assay performance in the same vessel have been introduced. 2. (ii) Time of hydrolysis is minimized. 3. (iii) The purification step has been omitted. The sensitivity of the method expressed as the lowest detectable level of hypro was estimated to 0.8 μg/ml of urine or bone sample. Samples ( n = 9) analyzed both by the present method and one published by Prockop and Udenfriend (2) exhibited a mean deviation of 3.5%. The mean error of duplicate analyses was 6.1% for urine samples ( n = 18) and 1.8% for bone samples ( n = 6). Hydroxyproline recoveries were 87.6 and 84.7% for urine and bone, respectively, and the interassay variation coefficients were 1.5% for authentic hydroxyproline standards and 3.0% for urine and bone samples. This method permits the daily assay of 40 urine samples in duplicate and can be easily adapted for bone tissue.