Background . The epidemiology of inborn errors of purine and pyrimidine (PnP) metabolism in developing countries is unknown. Facilities for the analysis of PnP metabolites in Malaysia are currently lacking owing to limited resources and expertise in this field. To achieve the correct diagnosis for these disorders is a time-consuming and costly process. The tests are not readily available for our seriously ill patients. Objective . The primary aim of this study was to establish a simple and cost-effective method using rapid-resolution high-performance liquid chromatography (RR-HPLC) for the diagnosis of inborn errors of PnP metabolism among Malaysian children who are suspected to have disorders of PnP metabolism. The secondary aim was to study the epidemiology and biochemical phenotype in our patients. Methods . The analytical method was developed using a reversed-phase high-performance liquid chromatography (RP-HPLC) with C18 column coupled to a diode array detector for simultaneous determination of the PnP metabolites. A total of 1499 patients were studied. 556 healthy children and adults were recruited as normal controls to establish age-related reference ranges and urinary uric acid – creatinine ratios. Results . The method established was able to separate up to 18 PnP metabolites in a single analytical run time of 28 minutes. Good precision (coefficient variation of 0.9993) was also observed. Recoveries were 99.8–108.4% for the tested metabolites. The detection limit of 2.18–12.5 μmol/l was adequate to detect patients with slightly increased concentration of these metabolites. Agerelated reference ranges among our population were established and were used for diagnostic interpretation related to this group of disorders. Twelve patients (0.8%) were diagnosed, including four with combined molybdenum cofactor deficiency, three with isolated sulphite oxidase deficiency, two with thymidine phosphorylase deficiency, one with adenylosuccinate lyase deficiency and two with dihydropyrimidine dehydrogenase deficiency. Four cases of urea cycle defects were also detected. Conclusions . The method that we developed was proven to be efficient, reliable and sensitive enough to be applied in our clinical laboratory for the diagnosis of inborn errors of PnP metabolism disorders among Malaysian children. Early recognition and correct diagnosis allowed prompt treatment, better outcome and genetic counselling.