Over the last two decades, misuse of 4-fluoroamphetamine (4-FA) became an emerging issue in many European countries. Stimulating effects last for 4-6 hours and can impact psychomotor performance. The metabolism of amphetamine-type stimulants is stereoselective and quantification of (R)- and (S)-enantiomers has been suggested for assessing time of use. To date no data on enantioselective pharmacokinetics is available for 4-FA in serum samples. An enantioselective liquid chromatography-tandem mass spectrometry (LC-MS-MS) method was developed using a chiral Phenomenex® Lux 3 μm AMP column. Validation of the method showed satisfactory selectivity, sensitivity, linearity (0.5-250 ng/mL), precision and accuracy. Recreational stimulant users orally ingested two doses (100 mg, n=12, and 150 mg, n=5) of 4-FA. Blood samples were drawn prior to application and over a period of 12 hours after ingestion and analyzed for 4-FA enantiomers. Peak concentrations and corresponding times did not differ significantly between the enantiomers (mean (R)/(S)-ratio at tmax 1.05, 0.85-1.16). With mean 12.9 (8.3-16.1) hours, apparent elimination half-lives (t1/2) were significantly (p < 0.01) longer for (R)-4-FA than for (S)-4-FA (6.0 hours; range 4.4-10.2 hours) and independent of the dose given. Over time, (R)/(S)-concentration-ratios were linearly increasing in all subjects to maximum ratios of 2.00 (1.08-2.77) in the last samples (after 12 hours). The slopes of the (R)/(S)-ratio exhibited marked inter-individual differences (0.023 to 0.157 h-1, mean 0.095 h-1). Ratios higher than 1.60 only appeared earliest after a minimum of 6 hours and therefore suggest the absence of acute drug effects. Different elimination half-lives of enantiomers lead to constantly increasing (R)/(S)-concentration-ratios. Consequently, ratios of 4-FA enantiomers in serum are a promising indicator for assessment of the time of drug consumption.