Urea and glucose are two important bio-markers in clinical chemistry, especially for the diagnosis of diabetes and renal failure.Serious diabetes patients usually have complication possibility of renal failure.Traditional methods of quantifying these analytes in serum involve complicated sample preparation and result in low sensitivity.To date, simultaneously determination these two bio-markers in serum samples at ng/ml (ppb) concentrations has not been reported.A high-throughput and ultra-sensitive method using normal-phase high performance liquid chromatography (HPLC) tandem triple quadrupole (QQQ) mass spectrometry was developed for the simultaneous determination of urea and glucose concentrations in serum.