In recent years, liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS) technique has been widely applied to analyze amino acids in biological fluids with protein precipitation or dilution of the sample.Because some amino acids with highly polar or charged side chains and the higher polar matrix load, the matrix effect in urine should be carefully assessed by using electrospray ion source mass spectrometry.Proper sample clean-up is therefore crucial.In order to diminish the matrix effect, a robust and sensitive HPLC-ESI-MS/MS method by coupling solid phase extraction (SPE) sample preparation was developed to analysis routinely quantified amino acids in urine sample.Optimization of SPE conditions was performed.The sample loading pH was critical for extraction efficiency.It was confirmed that 0.1M acetic acid (pH 2.8) had higher amino acids extraction efficiency.