Introducation: The use of Khat trend is increasing among higher education students in Jazan region. Immunoassay techniques are used in the poison control and forensic medical chemistry center in Assir region to screen the urine samples for the presence of amphetamine. Immunoassays are sensitive, fairly inexpensive and are relatively easy to perform. Poor specificity is a concern with the first step of the Urine Drug Screen (UDS) process, so a confirmatory test is recommended for immunoassay positive result. Aim of the work: This study was conducted to assess the cross-reacting effect of Cathinone and Cathine (the active principles of Khat) with amphetamine when analysed by immunoassay analysers and the efficacy of High Performance Thin Layer Chromatography (HPTLC) in differentiating between these chemicals and at what concentration. Materials and Methods: Twelve different concentrations from the standards of both Cathinone and Cathine were prepared. The samples were tested for Amphetamine Assay using two immunoassay analysers; Abbott AxSYM and Abbott Architect. The samples were then examined by HPTLC. Results: No cross-reactivity was detected on AxSYM while the samples examined by the Architect showed cross-reactivity at 5ug/ml for cathine and 70ug/ml for cathinone. Conclusion: HPTLC was unable to detect the difference between cathinone and cathine from Amphetamine at all prepared concentrations. Recommendations: We recommend the use of other confirmatory techniques such as; HPLC, GC-MS, or LC-MS, to examine all amphetamine positive samples by immunoassay analysers to detect the false positive samples and to detect the presence of Cathinone and Cathine especially in areas where Khat is frequently used.