Background: The enantiomers of the designer drugs 3,4-methylenedioxyamphetamine (MDA), 3,4-methylenedioxymethamphetamine (MDMA), and 3,4-methylenedioxyethylamphetamine (MDEA) differ in their pharmacologic and toxicologic potency. The aim of this study was to develop an assay for measuring these enantiomers in small plasma volumes and to analyze samples from a controlled study with MDMA. Methods: The analytes were extracted from 1.0 and increased over time. Concentrations of S-(+)-MDA exceeded those of R-(-).-MDA, their ratios (R vs S) also increasing over time but remaining <1.0. Conclusions: This assay enables sensitive, reliable, and fast enantioselective measurement of MDA, MDMA, and MDEA in small volumes of plasma. The controlled study data confirm previous findings of MDMA and MDA enantiomer ratios (R vs S) increasing over time after ingestion of racemic MDMA.