Abstract Background Semi-quantitative immunoassays (IA) are commonly used as the initial urine drug screen (UDS), with positive results confirmed by additional testing. However, substantial false negative (FN) rates have been reported in the classes of opioids and amphetamines. In our pediatric hospital, a semi-quantitative comprehensive urine drug testing by liquid chromatography quadrupole time-of-flight mass spectrometry (LC-QTOF) MS (referred as QTOF) is offered as either a direct order or a reflexive order following positive IA UDS. The lab also reflexes to run an IA UDS with every direct QTOF order. This study presents paired urine drug IA and QTOF results in the classes of opiate, synthetic opioid, and amphetamine/methamphetamine between 2019 and 2023 to understand trends and to evaluate assay accuracy. Methods Paired results from IA (Siemens Atellica) and QTOF (Sciex 4600) were analyzed with reporting cutoff concentrations from representative drugs in each class listed in Figure 1. The opiate class reported on QTOF includes 6-monoacetylmorphine, morphine, hydrocodone, codeine and metabolites. The synthetic opioid class includes buprenorphine, fentanyl, levorphanol, methadone, naloxone, oxycodone, tramadol and metabolites. Simultaneous findings of opiate and synthetic opioid were counted as the opiate class. FN rate of IA was calculated using QTOF as the reference. Results As shown in Figure 1, QTOF testing nearly doubled from 2019 to 2023, with an expected high FN rate in the synthetic opioid class and alarmingly rising FN rates in opiate and amphetamine classes. Within the synthetic opioid class, percentage of positive fentanyl results increased from 37.6% to 59.0% in 5 years, and ∼10% are only positive for norfentanyl, which is detected in very few commercial fentanyl IAs. Conclusions Our study clearly demonstrates the gap in pediatric toxicology investigation, reporting high FN rates in common drug classes by urine IA, calling for more comprehensive and sensitive UDS to be broadly adopted.