This study was to validate a LC-MS/MS method for the determination of 17 new synthetic opioids (NSOs) in hair including 3-fluorofentanyl, 3-methylfentanyl, acetylfentanyl, acetylnorfentanyl, alfentanyl, butyrylfentanyl, butyrylnorfentanyl, carfentanil, fentanyl, furanylfentanyl, furanylnorfentanyl, methoxyacetylfentanyl, norcarfentanil, norfentanyl, ocfentanil, sufentanil, and U-47700, and to apply it to 137 authentic samples. Twenty milligrams of hair was decontaminated in dichloromethane and underwent liquid extraction. 10 μL of the reconstituted residue were injected onto the system. The separation was performed in 12 minutes in a gradient mode at a flow rate of 300 μL/min using a Hypersyl Gold PFP column (100 × 2.1 mm i.d., 1.9 μm) maintained at 30°C. Compounds were detected in positive ionization and MRM modes using a TSQ Endura mass spectrometer (ThermoFisher). The method was validated according to EMA guidelines. The LLOQ was in the range 1-50 pg/mg, and the calibration ranged from the LLOQ-1000 pg/mg. Intra- and inter-day accuracy (bias) and precision were < 15%. Extraction recoveries of parent drugs and metabolites were 74-120% and 7-62%, respectively. The matrix effect was in the range 59-126% (CVs ≤ 12.9%). Fentanyl was found in six cases at concentrations of < 1-1650 pg/mg (n = 14 segments). Five fentanyl analogs were quantified in two cases: 3-fluorofentanyl (25-150 pg/mg, n = 5), furanylfentanyl (15-500 pg/mg, n = 5), methoxyacetylfentanyl (500-600 pg/mg, n = 2), acetylfentanyl (1 pg/mg, n = 2), carfentanyl (2.5-3 pg/mg, n = 2). This fully validated method allowed us to test for the first time 3-fluorofentanyl and norcarfentanil in hair among 15 other NSOs, and brings new data regarding 3-fluorofentanyl and methoxyacetylfentanyl hair concentrations.