A liquid chromatography - tandem mass spectrometry (LC-MS/MS) method has been developed to simultaneously measure four sodium glucose co-transporter 2 (SGLT2) inhibitors and the transfer marker antipyrine (ANTI) in perfusion medium and placental tissue collected from ex vivo human placental perfusions. The four SGLT2 inhibitors were empagliflozin (EMPA), dapagliflozin (DAPA), ertugliflozin (ERTU) and canagliflozin (CANA). Chromatographic separation was achieved on an Uptisphere® C18 reversed phase column (50 mm × 4.6 mm × 5 µm) within 2.85 min, using a gradient elution with 10 mM ammonium formate in water (mobile phase A) and acetonitrile (mobile phase B) both with 0.1% formic acid. Analysis of ammonium adduct ions was performed on an AB SCIEX 6500+ triple quadrupole mass spectrometer using positive electrospray ionisation and scheduled multiple reaction monitoring (sMRM). The transitions were m/z 468.00 → 355.20 (EMPA), m/z 426.00 → 167.20 (DAPA), m/z 437.10 → 206.90 (ERTU), m/z 462.00 → 249.00 (CANA) and m/z 189.20 → 55.90 (ANTI). The method was validated according to the European Medicines Agency guidelines and was proven to be selective, linear within a concentration range of 1-1000 µg/L (DAPA, CANA, ANTI) and 1-500 µg/L (EMPA, ERTU), accurate, precise and free of carry-over, instabilities, recovery and matrix effect issues. This newly developed method is suitable to analyse perfusion medium and placenta tissue samples collected during ex vivo human placenta perfusions. It thereby enables quantification of transport across the placental barrier of the SGLT2 inhibitors EMPA, DAPA, ERTU and CANA as well as the transfer marker ANTI