This paper describes a fully automated online method for solid-phase extraction coupled with liquid chromatography and tandem mass spectrometry using atmospheric pressure ionization (LC-LC-APPI-MS/MS) to simultaneously detect selected dissolved natural and synthetic hormones at concentrations as low as 5 ng/L from aqueous matrices. The method shows excellent performance for the direct analysis of water and wastewater with respect to calibration curve linearity, analytic specificity, sensitivity, and carryover, as well as overall method accuracy and precision. With a runtime of 15 min, a minimum of 200 samples were analyzed using a single online solid-phase extraction (SPE) column without noticeable differences in system performance. Because of the ruggedness and simplicity of this system, generic methods can be easily developed and applied for the high-throughput analysis of a wide variety of compounds without the need to resort to laborious offline SPE sample preparation.