Collagen is the major structural protein of the extracellular matrix in many tissues. Hydroxyproline, a major component of collagen, comprises around 13.5% of its amino acid composition. Due to its highly restricted distribution in collagen, the hydroxyproline content accurately reflects the amount of collagen in the sample. Therefore, quantitating hydroxyproline has been utilized for evaluating tissue fibrosis or collagen deposition (1, 2, 3). However, classic hydroxyproline assays are not useful since it requires cumbersome procedures and special tools. This hydroxyproline assay kit employs an improved assay system that can be operated with ease and precision using 96-well plates. This kit works for quantitation of total collagen of any type and species in tissue specimens and tissue homogenates.