The use of corticosteroid prodrugs in pharmacokinetic 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 the anticoagulant EDTA as a stabilizer for dexamethasone sodium phosphate (DSP) in rat plasma and provides simultaneous HPLC analysis of DSP and dexamethasone. An already developed ion-paired reversed-phase HPLC assay for simultaneous measurement of corticosteroid phosphate ester prodrugs and their active steroids was applied in this study. This assay was used for analyzing samples from an in vitro DSP hydrolysis study in rat plasma. In agreement with allometric principles, the prodrug hydrolysis occurred at a much faster rate (in vitro half-life of 1.75 h) in rat plasma as compared with previously reported prodrug hydrolysis half-life of 10-12 h in sheep and human plasma. The in vitro degradation of the prodrug in rat plasma was greatly minimized in plasma containing EDTA at the concentration commonly used an anticoagulant. This study demonstrates that artifacts in pharmacokinetic profiles of corticosteroids due to in vitro prodrug hydrolysis can be greatly minimized by collecting blood samples with EDTA as the anticoagulant.