Inborn errors of purine metabolism, either deficiencies of synthesis or catabolism pathways, lead to a wide spectrum of clinical presentations: urolithiasis (adenine phosphoribosyltransferase), primary immune deficiency (adenosine deaminase deficiency and purine nucleoside phosphorylase deficiency), severe intellectual disability, and other neurological symptoms (Lesch-Nyhan disease, adenylosuccinase deficiency, and molybdenum cofactor deficiency). A rapid quantitative purine assay was developed using UPLC-MS/MS to determine purine nucleoside and base concentrations in urine. Taking advantages of ultra-performance liquid chromatography, we achieved satisfactory analyte separation and recovery with a polar T3 column in a short run time with no requirement of time-consuming sample preparation or derivatization. This targeted assay is intended for diagnosis and management of purine diseases, newborn screening follow-up of SCID, and evaluation of autism spectrum disorders.