This work presents the validation of a new immunological assay, the One-Step enzyme-linked immunosorbent assay (ELISA) tests from International Diagnostic Systems Corp. for the screening of drugs of abuse (cannabis, amphetamines, opiates, and cocaine) in human hair, with subsequent GC-MS confirmation. After decontamination and segmentation into small pieces, 50 mg of hair sample were incubated in 1 ml of methanol during 16 h at 40 degrees C. A 100 microL aliquot was collected and evaporated to dryness in presence of 100 microL of methanol/hydrochloric acid (99:1, v/v) to avoid amphetamines loss. The dried extract was dissolved in 100 microL of the "sample and standard diluent" solution included in the kit. This solution was submitted to analysis according to the recommended instructions of the manufacturer. During the validation phase, GC-MS confirmations were conducted according to our fully validated and published methods for opiates, cocaine, cannabis, and amphetamines determinations in hair. In a last development step, these procedures were slightly modified to directly confirm ELISA results by GC-MS using the methanolic extract. Ninety-three specimens were simultaneously screened by the ELISA tests (103 for tetrahydrocannabinol (THC)) and confirmed by GC-MS. Twenty were found positive for cannabis (THC: 0.10-6.50 ng/mg), 21 for cocaine (0.50-55.20 ng/mg), 24 for opiates (6-acetylmorphine (6-AM): 0.20-11.60 ng/mg, MOR: 0.20-8.90 ng/mg, codeine (COD): 0.20-5.90 ng/mg), and 13 for amphetamines (AP: 0.20 and 0.27 ng/mg, methamphetamine (MAP): 0.30 and 1.10 ng/mg, methylenedioxymethamphetamine (MDMA): 0.22-17.80 ng/mg). No false negative results were observed according to the Society of Hair Testing's (SoHT) cutoffs (0.5 ng/mg for cocaine, 0.2 ng/mg for opiates and amphetamines, and 0.1 ng/mg for THC). The One-Step ELISA kits appear suitable due to their sensitivity and specificity for drug of abuse screening in hair. This technology should find interest in workplace drug testing or driving license regranting, especially when many samples have to be tested with a high rate of negative samples, as ELISA is an easy and high-throughput method.