A procedure is presented for proline quantification in serum filtrates and urine hydrolysates. Interference from primary amino acids is greatly reduced by nitrozation. After nitrozation and neutralization, proline is reacted with ninhydrin in a concentrated electrolyte solution. The resulting red proline—ninhydrin product is salted out, extracted into benzene, and measured at 520 nm. The extent and nature of interference from various amino acids have been determined, as has the effect of urinary constituents. Procedural variables have also been studied. Preliminary data are presented for urinary proline/creatinine ratios for 6-to 12-month-old infants.