The determination of purines and pyrimidines in biofluids is useful for the clinical-biochemical characterization of acute and chronic pathological states that induce transient or permanent alterations of metabolism. In particular, the diagnosis of several inborn errors of metabolism (IEMs) is accomplished by the analysis of circulating and excreted purines and pyrimidines. It is certainly advantageous to simultaneously determine the full purine and pyrimidine profile, as well as to quantify other compounds of relevance (e.g., organic acids, amino acids, sugars) in various metabolic hereditary diseases, in order to screen for a large number of IEMs using a reliable and sensitive analytical method characterized by mild to moderate costs. Toward this end, we have developed an ion-pairing HPLC method with diode array detection for the synchronous separation of several purines and pyrimidines. This method also allows the quantification of additional compounds such as N-acetylated amino acids and dicarboxylic acids, the concentrations of which are profoundly altered in different IEMs. The application of the method in the analysis of biological samples from patients with suspected purine and pyrimidine disorders is presented to illustrate its applicability for the clinical-biochemical diagnosis of IEM.