AbstractIn recent times, structural variants of fentanyl (designer fentanyls) have appeared on the recreational drug market for new psychoactive substances (NPS). These potent opioids have caused harmful intoxications and increased opioid‐related mortality in many countries. This work evaluated 3 commercial immunoassays for fentanyl screening in urine and investigated whether they are useful also for screening of designer fentanyls. The assays examined were the Thermo DRI® Fentanyl Enzyme Immunoassay, the ARK™ Fentanyl Assay homogeneous enzyme immunoassay, and the Immunalysis® Fentanyl Urine SEFRIA™ Drug Screening Kit. A liquid chromatography–high‐resolution mass spectrometry method was used as reference. The DRI fentanyl immunoassay generated somewhat higher assay imprecision values (%CV) compared with the ARK™ and SEFRIA™ assays, but all assays showed %CV values acceptable for routine use. The 3 assays showed overall good detectability (33%–95% cross‐reactivity) for blank urine samples spiked with acetylfentanyl, acrylfentanyl, butyrfentanyl, 4‐chloroisobutyrfentanyl, 4‐fluorobutyrfentanyl, 4‐fluorofentanyl, 4‐fluoroisobutyrfentnyl, isobutyrfentanyl, methoxyacetylfentanyl, or tetrahydrofuranfentanyl, whereas 4‐methoxybutyrfentanyl (all assays) and 2‐fluorofentanyl (DRI assay) showed low cross‐reactivity. A good detectability of designer fentanyls was confirmed in urine samples from authentic acute intoxications. In conclusion, the present results demonstrate that the urinary fentanyl immunoassays are generally useful also for preliminary screening of fentanyl analogs sold as NPS. When the SEFRIA™ assay was applied for testing of 980 urine samples from patients treated for drug dependence in Sweden, only 1 sample was confirmed positive for fentanyl.