It is rather difficult to determine simultaneously more than one drug from different biological samples and to interpret the clinical picture in the case of multiple drug abuse. All mentioned drugs are life-threatening solely and the risk is becoming higher when they are used together. In this study, a gas chromatography-mass spectrometry (GC-MS) method was developed and validated for the simultaneous detection and quantification of amphetamine (AMP), 3,4-Methylenedioxymethamphetamine (MDMA) and cocaine in serum sample. The analytes were extracted by solid-phase extraction (SPE) cartridges, derivated with heptafluorobutyric anhydride at 60 degrees C for 30 min and analyzed by GC-MS. Correlation coefficient values were found satisfactory. Linearity ranged from 1 to 20 mu g/mL for all three substances. After acceptable results were obtained from calibration solutions, the study was conducted to observe matrix-match calibration and linearity parameters. Extraction efficiency of the method was observed in two concentrations spiked to serum matrix (5 and 10 mu g/mL) and found over 80% for three illicit drugs. Precision, accuracy, stability evaluation were appropriate as well. The validated analytical method was applied to an authentic sample which obtained from a patient administered to emergency room. The patient was suspected in regard to multi-drug use of stimulants and serum sample was analyzed by the presented method and AMP, MDMA and cocaine use was determined with concentrations of 0.45, 1.03 and 1.98 mg/mL in blood respectively. The developed method proved capable of quantifying simultaneous AMP, MDMA and cocaine use proposed in this study. Multidrug abuse is not a novel phenomenon but has to be determined as soon as possible by forensic toxicology laboratories because of potential severe health risks.