The analysis of corticosteroid prodrugs in pharmacokinetic (PK) studies poses the risk of overestimation of corticosteroid concentrations due to in vitro hydrolysis of prodrugs after sample collection. This study tests the effectiveness of enzyme inhibitors as stabilizers for betamethasone sodium phosphate (BSP) in pregnant sheep plasma samples collected during PK studies with betamethasone (BET) and provides simultaneous high-performance liquid chromatography analysis of BSP and BET. A rapid, sensitive, and specific ion-paired reversed-phase high-performance liquid chromatography assay for simultaneous measurement of BET and BSP in plasma was developed. This assay was used for analyzing samples from an in vitro prodrug hydrolysis study. Enzyme inhibitors tested were sodium arsenate (Na 2 HAsO 4 ) and ethylenediaminetetraacetic acid. The BSP was administered intramuscularly to three pregnant sheep to assess in vivo PK. Samples were split with part treated with Na 2 HAsO 4 and part left natural. In vitro hydrolysis of BSP in plasma to BET could be completely inhibited by Na 2 HAsO 4 , but not by ethylenediaminetetraacetic acid. The PK study showed lower concentrations of BET in samples with Na 2 HAsO 4 compared with natural samples. This study demonstrates that artifacts in PK profiles of corticosteroids due to in vitro prodrug hydrolysis can be prevented by sample treatment with enzyme inhibitors.