Following the green analytical chemistry principles, an efficient strategy involving second-order data provided by liquid chromatography (LC) with diode array detection (DAD) was applied for the simultaneous determination of estriol, 17β-estradiol, 17α-ethinylestradiol and estrone in natural water samples. After a simple pre-concentration step, LC-DAD matrix data were rapidly obtained (in less than 5 min) with a chromatographic system operating isocratically. Applying a second-order calibration algorithm based on multivariate curve resolution with alternating least-squares (MCR-ALS), successful resolution was achieved in the presence of sample constituents that strongly coelute with the analytes. The flexibility of this multivariate model allowed the quantification of the four estrogens in tap, mineral, underground and river water samples. Limits of detection in the range between 3 and 13 ng L−1, and relative prediction errors from 2 to 11% were achieved.Fil: Pérez, Rocío Laura. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Química Rosario (IQUIR-CONICET); Argentina.Fil: Escandar, Graciela Mónica. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Química Rosario (IQUIR-CONICET); Argentina.