A growing concern has been raised about human exposure to phthalates and bisphenols, while data is limited regarding the accumulation of these endocrine disrupting chemicals at the target tissue. In this study, a novel, simple, and sensitive method was successfully developed for the simultaneous determination of nine phthalate monoesters and nine bisphenols in human prostate tissue samples. A solid-liquid extraction procedure was applied following ultra-high performance liquid chromatography tandem mass spectrometry analysis. The detection and quantification limits were in the range of 4.12 × 10-3 to 0.370 ng/g and 1.38 × 10-3 to 1.23 ng/g, respectively. The average spiked recoveries varied from 71.4% to 102%, with relative standard deviations ≤ 10%. Finally, this method was applied to 76 human prostate tissue samples. Four phthalate monoesters (mono-ethyl phthalate, mono-iso-butyl phthalate, mono-n-butyl phthalate, and mono-benzyl phthalate) were detected with the highest frequency of 98.68%, followed by other five phthalate monoesters (71.05% ∼ 94.74%), bisphenol S (72.37%), and bisphenol A (57.89%). Five bisphenols were not detected in any tissue sample. The concentrations of detected phthalate monoesters and bisphenols ranged from 1.12 × 10-3 to 1.86 × 102 ng/g and 6.08 × 10-3 to 39.0 ng/g, respectively, with standard errors ranging from 3.25×10-3 to 4.64 ng/g. Besides, a positive correlation for the concentration of seven phthalate monoesters and bisphenol A could be observed between tissue and urine, which indicates that these metabolites in urine can serve as noninvasive biomarkers to evaluate the true exposure level of prostate tissue. This study provides data and information on exposure to phthalate monoesters and bisphenols in human prostate tissue and the association with their urinary metabolites, supporting further studies of pollutant exposure and prostate disease.© 2025 Wiley‐VCH GmbH.