To identify chemicals with endocrine-disrupting activity easily, we developed a new bioassay system, consisting of bioassay using genetically modified yeast expressing human estrogen receptor and high performance liquid chromatography (HPLC), in which advantages of instrumental analysis and bioassay are combined. The peaks in the mixture of these estrogen-like compounds analyzed using an HPLC bioassay were similar to those obtained by analysis using an HPLC-UV detector. Underground water and sea sediment were analyzed by an HPLC bioassay, and detected a few estrogen-like compounds, respectively. Estrogen-like compounds and yeast-growth inhibitors can be separated by HPLC-bioassay.