Abstract Background Urine drugs of abuse testing (DOAU) in US hospitals are mainly orders from the emergency department performed by immunoassay screening. Testing can be relatively expensive, and results may suffer from non-idealities in sensitivity, specificity, and from panel limitations. A potential approach to these issues for immunoassays is instead to employ mass spectrometry (MS), where a large and flexible analyte list can be evaluated providing needed specificity and reducing send out costs for confirmatory testing. We compared results and cost impacts of a DOAU assay on a prototype, ion mobility mass spectrometry system to those obtained from an array of immunoassays offered as routine tests at a large urban hospital. Methods Immunoassay screen-positive and screen-negative urine samples were isolated from among those submitted for a DOAU panel (amphetamines, barbiturates, benzodiazepines, cannabis, cocaine, opiates, and methadone) or for additional single drug tests (fentanyl, oxycodone, phencyclidine, buprenorphine, propoxyphene). Sample aliquots (3 mL) were retrieved after not more than 5 days of refrigerated storage of the original sample and were subsequently frozen. These samples were then subjected to MS analysis using the prototype system where identification of 62 compounds was based on mass and ion mobility-derived Collision Cross Section (CCS). An established library of m/z, SLIMCCSHe, peak intensity, and peak width for each analyte was used, with acceptable method performance characteristics as based on applicable CLIA validation guidelines. The MS list included compounds covered by the immunoassay classes as well as additional drugs not expected to be positive among the immunoassays test results but of value for routine toxicological assessments. Results comparisons, utility of results, sample-to-result turnaround times, and overall financial implications were compared between the two workflows. Results Among immunoassay screen-negative samples, MS analysis showed additional positive results for metabolites of parent drugs as well as novel psychoactive substances and known adulterants in >10% of cases. Among the immunoassay screen-positive results, MS analysis confirmed immunoassay results (>90%) but picked up an additional positive component in over 15% of cases. Cost per complete patient result for the immunoassay was approximately $2.80 in comparison to an estimated cost of $5 for the MS method. Confirmation testing for positive immunoassay screen results (16% of all samples) would cost an additional $70 per sample, whereas the need for confirmation testing is eliminated for the MS method. Thus, use of the MS testing workflow would be superior to an immunoassay with reflex testing workflow while at the same time providing a clinically superior service of more diverse positive test results and elimination of often overlooked false negatives. Turnaround time between immunoassay (1h) and MS (50m) were judged to be similar. Conclusions The MS approach confirmed >90% of all immunoassay positive screen results. In addition, the MS method identified additional positive results not detected by the immunoassay despite an overall similar estimated time for sample to patient. Need for development of a user friendly, MS analyzer for use in hospitals is supported based on cost-effective and analytically superior tests for DOAU compared to screens performed using immunoassay panels.