Objective To establish a comprehensive analytical method based on SPE-UPLC-MS for the simultaneous determination of bisphenol A (BPA), nonylphenol (NP), and octylphenol (OP) in urine samples.Methods Sixty urine samples collected from healthy subjects were analyzed for BPA, NP, and OP concentrations. The samples were de-conjugated by adding β-glucuronidase and sulfatase. After the enzymatic treatment, the samples were subjected to the OASIS HLB column solid phase extraction cartridges so as to be cleaned and concentrated. The UPLC separation was performed on a Acquity UPLCTM BEH C18 column (2.1x100 mm, 1.7 μm) with a gradient elution system of methanol-water as the mobile phase. Triple-quadrupole mass spectrometry analyzer was used for the qualitative and quantitative analysis of UPLC-MS/MS system.Results The limit of detection of BPA, NP, and OP was 0.10, 0.10, and 0.15 ng/mL, respectively. The recoveries of BPA, NP and OP were 80.1%-108%, 81.3%-109%, and 81.5%-98.7%, respectively. Among the 60 urine samples, BPA was detected in 8 samples at the level of 0.297-32.7ng/mL, NP was detected in 29 samples at the level of 1.69-27.8 ng/mL, and OP was detected in 17 samples at the level of 0.407-11.1 ng/mL.Conclusion The method is simple with high sensitivity and selectivity, and is suitable for the determination of BPA, NP, and OP in urine. As shown by our analysis, BPA, NP, and OP appear to be prevalent in human urine. This is particularly true for NP. The results from our study is therefore valuable for future studies to assess the exposure to BPA, NP, and OP in the general population.