The increasing demand to monitor multiple classes of analytes has been mirrored by increased analytical cost and decreased throughput. For instance, the analyses of estrogens and antibiotics by liquid chromatography with tandem mass spectrometry (LC-MS/MS) are typically performed in two separate methods because estrogen analysis requires electrospray with negative ionization, while sulfonamide and tetracycline antibiotics are analyzed under positive ionization. Therefore, we investigated the use of wrong-way-round (WWR) ionization to demonstrate that sulfonamides and tetracyclines can be analyzed at a high pH (10.4), allowing simultaneous analysis with free and conjugated estrogens. An LC-MS/MS method was developed for 28 compounds by polarity switching, based on WWR ionization brought about by the ability of ammonium ions to protonate basic compounds in the gas phase even at high pH. Mass spectral data suggest that gas-phase chemical ionization induced by ammonium ions to form adducts [M + NH(4)](+) occurred, with the subsequent dissociation to the molecular ion [M + H](+). Almost all compounds have an increased signal-to-noise (S/N) ratio of [M + H](+) for sulfonamides and tetracyclines when ionized in basic versus acidic mobile phases by direct injection (no column), indicating that detection limits were not compromised. This study demonstrates a successful application of WWR ionization for the simultaneous analysis of multiple classes of compounds in a single LC-MS/MS analysis.