The method presented in this study demonstrates the use of Ostro Sample Preparation Plates combined with UPLC/MS/MS for the analysis of 22 opioid compounds and metabolites of interest in whole blood samples. All compounds were analyzed in less than 5.5 minutes with complete resolution of all isobaric compound pairs. The use of Ostro Sample Preparation Plates allowed for rapid, in-well cell lysis and protein precipitation, followed by elution into a 96-well collection plate. This procedure resulted in improved throughput compared to protein precipitation in individual tubes, with the added benefit of removing endogenous phospholipid compounds. All analytes demonstrated good linearity over the entire calibration range, and the method was sensitive for reliable detection at the lowest curve points.