The primary objective of this study is to validate a new fast method for determination of uric acid in milk. The method is based on an enzymatic-fluorometric technique that requires minimal pre-treatment of milk samples. The present determination of uric acid is based on the enzymatic oxidation of uric acid to 5-hydroxyisourate via uricase where the liberated hydrogen peroxide reacts with 10-acetyl-3,7-dihydroxyphenoxazine via peroxidase and the fluorescent product, resorufin, is measured fluorometrically. Fresh composite milk samples (n=1,072) were collected from both Jersey (n=38) and Danish Holstein (n=106) cows from one local herd. The average inter- and intra-assay variations were 7·1% and 3·0%, respectively. Percent recovery averaged 103·4, 107·0 and 107·5% for samples spiked with 20, 40 or 60 μm of standard, respectively, with a correlation (r=0·98; P<0·001) observed between the observed and expected uric acid concentrations. A positive correlation (r=0·96; P<0·001) was observed between uric acid concentrations using the present method and a reference assay. Storage at 4°C for 24 h resulted in lower (P<0·01) uric acid concentrations in milk when compared with no storage or samples stored at −18°C for 24 h. Addition of either allopurinol (a xanthine oxidase inhibitor) or dimethylsulfoxide (a solvent for allopurinol) did not affect milk uric acid concentrations (P=0·96) and may indicate that heat treatment before storage and analysis was sufficient to degrade xanthine oxidase activity in milk. No relationship was observed between milk uric acid and milk yield and milk components. Authors recommend a single heat treatment (82°C for 10 min) followed by either an immediate analysis of fresh milk samples or storage at −18°C until further analysis.