A selective and highly sensitive analytical methodology for determination of Bisphenol A in human plasma was developed and validated. The method was based on selective liquid/solid extraction, combined with liquid chromatography–electrospray ionization tandem mass spectrometry in the multiple reaction monitoring mode and negative ionization. The linearity of the detector response was verified in human plasma over the concentration range 0.100–200 ng mL−1. The detection limit was 0.03 ng mL−1 and the quantification limit was 0.100 ng mL−1. The analytical features of the proposed in-house validated method were satisfactory: precision was <10% and recoveries were around 84–104%. The matrix effect was studied and compensated using deuterated labeled standard. The applicability of the proposed method was demonstrated analyzing human plasma samples from individuals affected by non-alcoholic fatty liver disease. Bisphenol A was detected above the detection limit in all samples. The data show a persistence of unconjugated Bisphenol A levels in plasma and indicate a chronic Bisphenol A exposure of the target organ, suggesting an association between liver health status and Bisphenol A exposure. The results from our study are valuable for further investigation with large sample size and longitudinal study designs, necessary to confirm the observed association.