Progesterone concentrations are routinely monitored during in vitro fertilization cycles. Immunoassay-based platforms are used most often in this setting because they are simple to use and amenable to same-day sample collection and result-reporting. However, immunoassay methods are subject to variation in specificity between different assay manufacturers. In this study, a set of unexpectedly high progesterone concentrations led to a method comparison between two in-house immunoassay platforms relative to the reference method. Progesterone was measured in 28 serum samples from women undergoing IVF cycles using the Siemens ADVIA Centaur Immunoassay system and the Abbott Architect i1000SR analyzer. A subset of these samples was selected for progesterone measurement by liquid chromatography-tandem mass spectrometry to define the accuracy of each immunoassay. The Siemens ADVIA Centaur immunoassay system overestimated progesterone concentrations by 19% and the Abbott Architect overestimated progesterone concentrations by 5%. The Abbott Architect progesterone immunoassay provides a more accurate measurement of serum progesterone than the Centaur immunoassay at concentrations relevant for monitoring in vitro fertilization populations.