A high-performance liquid chromatography method for the analysis of allantoin, uric acid, hypoxanthine and xanthine in urine of sheep is described. Urine samples were analyzed directly after dilution. The combination of HPLC reversed phase C 1 8 columns with monitoring the effluent at 205 nm provides a relatively rapid and simple analytical tool for studying purine derivatives in the urine of ruminants. Separation and quantification of purine derivatives was achieved using three C₁₈ columns connected in a series together with a NH₄H₂PO₄ - NH₄H₂PO₄ and methanol (95:5) gradient. The average recoveries of standard compounds added to urine samples were satisfaction (96-103%). The low value of the intra-assay coefficient of variation and high recovery point to the satisfactory precision and accuracy of the reported method.