BACKGROUND Amino acids, purines and derivatives play a key role in physiological and pathological processes, such as the development of gestational diabetes mellitus. However, little literature has reported the simultaneous quantification of amino acids and purines. Therefore, a reliable and robust method for the determination of amino acids and purines is necessary. METHODS A rapid and simple method based on ultrahigh-performance liquid chromatography/tandem mass spectrometry (UHPLC/MS/MS) was developed to determine the concentrations of twenty amino acids or derivatives and three purines or derivatives in serum. RESULTS The method was validated for linearity of calibration curve, limit of detection, limit of quantification, inter-day and intra-day precision, recovery, stability and matrix effect. The correlation coefficients of calibration curves were higher than 0.993 except for isoleucine. The recoveries ranged from 78.4% to 128.0% for three different concentrations of the spiked analytes. The matrix effect value ranged from -7.3% to 9.2%. CONCLUSIONS A rapid and simple UHPLC/MS/MS method for the simultaneous measurement of amino acids, purines and derivatives in serum samples was developed. The method was successfully applied to determine and compare the concentrations of analytes between gestational diabetes mellitus samples and controls.