Cytochrome P450 (CYP) constitutes a superfamily of heme- containing enzymes that catalyze the oxidative biotransformation of structurally diverse xenobiotics including pharmaceuticals and drugs. Cytochrome P450 3A4 is not only the most abundant isoform in human liver but is also responsible for metabolizing approximately 60% of therapeutic drugs. This feature makes CYP3A4 highly susceptible to both reversible and irreversible, such as mechanism-based, inhibition. Mechanism-based inhibition is characterized by being time dependent as well as NADPH and concentration dependent, when some drugs are converted by CYPs to reactive metabolites. The inactivation of CYP3A4 can be due to chemical modification of the heme, the protein, or both by covalent binding of modified heme to the protein. Compared to reversible inhibition, mechanism-based inhibition of CYP3A4 more frequently causes unfavourable drug- drug interactions (DDI), as the inactivated CYP3A4 has to be replaced by newly synthesized CYP3A4 protein. DDI can lead to higher exposure of co-administered drugs, sometimes leading to toxicity. For these reasons, drug metabolism groups within pharmaceutical companies need a well established screening assay to assess mechanism-based inactivation of major human P450 enzymes by new chemical substances that are being developed by the company. Historically, adverse drug interactions were found in clinical trials or after the drugs were commercially released. That caused pharmaceutical companies large economical losses, since a large portion of development cost was in vain.Medivir AB is a small pharmaceutical company that would like to set up a screening method to be used to test candidate drugs for CYP3A4 mechanism-based inhibition. The central aim of this master degree project was to set up a screening assay to test irreversible inhibition of CYP3A4 and validate it with known inhibitors. Using the validated assay, a number of in-house project compounds will be measured for inhibition potential and the results analyzed for structure-activity correlations. Relationships between some functional groups and mechanism-based inhibition can provide insight for improvement of drug candidates and inhibition liability. The assay was based on microsomes containing recombinant human CYP3A4 and activity measured by conversion of the substrate dibenzylfluorescein into a fluorescent product. The product was quantified by measurement of fluorescence in a 96-well plate reader. Optimization was achieved by determining the reaction linearity with time and enzyme concentration. When possible the Km for the probe substrate was also determined. The effect of different backgrounds was studied to settle on a compensation for the enzyme activity. The effect of DMSO on CYP3A4 mediated metabolism of the substrate was studied to determine the acceptable solvent concentration. The concentration responsible for 50% inhibition (IC50) was also determined for several known inhibitors and compared with literature data.