Bisphenol A (BPA) is an environmental endocrine-disrupting chemicals that is widely used in common consumer products. There is an increasing concern regarding human exposure to BPA owing to the potential adverse effects associated with its estrogenic activity. For assessing environmental exposure to BPA, it is essential to have a sensitive, accurate and selective analytical method, especially one that can detect low BPA levels in complex sample matrices. In this study, we developed and validated an accurate, sensitive, and robust liquid chromatography-tandem mass spectrometry method for simultaneous quantification of free BPA and BPA -d-glucuronide (BPA-gluc) concentrations in human urine with only a single injection. Calibration curves were linear over a concentration range of 1-100ng/mL for BPA and 10-1000ng/mL for BPA-gluc. The levels of the analytes were determined quantitatively with HPLC/ESI-MS/MS by using negative electrospray ionization in the select ion monitoring mode and a pentaflouraphenyl propyl column. The validated method was applied to the analysis of spot urine specimens collected from randomly selected healthy human subjects. Copyright (c) 2013 John Wiley & Sons, Ltd.