Appropriate biomarkers of human exposure are required for epidemiological studies of endocrine disruption. We addressed this issue by improving a standardized method to assess the total effective xenoestrogen burden (TEXB), a biomarker of xenoestrogen exposure. Extensive separation of xenoestrogens from endogenous hormones was made in 20 adipose tissue samples by HPLC, and two eluates were separated and tested in the E-Screen bioassay. An extensive fractionation protocol was also developed. The objective of this study was to investigate predictors of TEXB by using a multiple regression model after adjusting by confounding factors. The final model included the estrogenicity of 8 out of 11 individual 1-min fractions into which the xenoestrogen eluate was split and the marital status of patients, and it explained 97% of TEXB variability, and variables. Our results indicate that TEXB of complex mixtures can be accurately predicted from the estrogenicity of a small number of components.