BackgroundImmunoassays provide simple, powerful and inexpensive screening methods for urine drug screening. Other substances and/or factors may interfere with the test and cause false or positive results. It is essential to understand the differences between methods to be able to evaluate their impact on the results. All evaluated immunoassays were assessed in comparison with GC–MS or LC-MS/MS, which are generally accepted as vigorous confirmation gold standard techniques. MethodsCEDIA, DRI, EIA and EMIT II Plus screening immunoassays were evaluated on Beckman-Coulter AU5810 analyser. All results were confirmed using GC–MS or LC-MS/MS methods. Measurement Uncertainty for immunoassays was calculated by using standard deviation multiplied by 1.96 to cover 95% confidence interval of tested samples. ResultsNo discrepancy was found between CEDIA and EMIT II Plus for cocaine, methadone, heroin, and benzodiazepines assays. No discrepancy was found between oxycodone DRI assay and Immunalysis enzyme immunoassay (EIA). Cannabinoids EMIT II Plus assay performed better than DRI assay. EMIT II Plus assays for amphetamine and ecstasy performed better than CEDIA amphetamine/ecstasy combined assay. Opiates EMIT II Plus assay performed better than CEDIA assay. Fentanyl Ark EIA method performed slightly better than the DRI method. Buprenorphine CEDIA second-generation assay performed better than CEDIA first-generation assay, EMIT II Plus assay and EMIT II Plus with added Beta-Glucuronidase assay. Measurement Uncertainty for immunoassays was calculated and tabulated. ConclusionsThis study covered a fundamental gap in available knowledge by evaluating the performance of screening the current new generation of immunoassays methods for drugs of abuse in urine against gold standard methods.