A significant number of people worldwide consume khat on daily basis. Long term of khat chewing has shown negative impact on several organ systems. It is likely that these people are co-administered khat preparations and conventional medication, which may lead to khat-drug interactions. This study aimed to reveal the inhibitory potencies of khat ethanol extract (KEE) and its major active ingredient (cathinone) on human cytochrome P450 (CYP) 2C9, CYP2D6, and CYP3A4 enzymes activities, which are collectively responsible for metabolizing 70-80% clinically used drugs.In vitro fluorescence-based enzyme assays were developed and the CYP enzyme activities were quantified in the presence and absence of KEE and cathinone employing Vivid CYP450 Screening Kits.KEE inhibited human CYP2C9, CYP2D6, and CYP3A4 enzyme activities with IC of 42, 62, and 18 μg/ml. On the other hand, cathinone showed negligible inhibitory effect on these CYPs. Further experiments with KEE revealed that KEE inhibited CYP2C9 via non-competitive or mixed mode with K of 14.7 μg/ml, CYP2D6 through competitive or mixed mode with K of 17.6 μg/ml, CYP3A4 by mixed inhibition mode with K of 12.1 μg/ml.Khat-drug interactions are possible due to administration of clinical drugs metabolized by CYP2C9/CYP2D6/CYP3A4 together with khat chewing. Further in vivo studies are required to confirm our findings and identify the causative constituents of these inhibitory effects.