Background: In Egypt, incidents of road and train accidents caused by drivers under the influence of drugs have led to the introduction of a law that mandates drug testing for public sector employees. Aim of the work: This study aimed to explore various methods for the detection of subversion of 11-nor-9-carboxy-D9-tetrahydrocannabinol (THC–COOH) and tramadol-positive urine samples. Subjects and Methods: After confirming the negativity and positivity of urine samples for THC–COOH and tramadol by using GC-MS, the urine samples were divided into negative control, THC–COOH-positive, and tramadol-positive samples. Positive samples were further sub-divided into six groups, one group without adulterants and five groups were adulterated with carbonated water, laundry detergent gel, tea, hydrogen peroxide, and drinking water at 20% and 40% concentrations (conc.) in each subgroup. Results: The integrity strips showed normal pH, specific gravity (SG), and negative nitrites that were corroborated by a pH meter and refractometer. As well as the creatinine levels were in the reference ranges. The rapid drug immunoassay strips yielded false-negative results for THC–COOH in all adulterated and diluted urine samples at both concentrations. Meanwhile, the 40% concentration of urine samples manipulated with tea, hydrogen peroxide, and drinking water displayed false negative results for tramadol. Conclusion: THC–COOH and tramadol-positive urine samples adulterated with at 20%, 40% conc. of laundry detergent gel and 40% conc. of tea displayed false negative results by automated immunoassay and GC-MS. The automated immunoassay provided accurate results for drug screening of most adulterated and diluted urine samples that tested positive for THC–COOH and tramadol compared with GC-MS.