In this study we describe a battery of fluorescence assays for rapid measurement in intact cells of the activity of nine cytochromes P450 (P450s) involved in drug metabolism. The assays are based on the direct incubation of monolayers of cells expressing individual P450 enzymes with a fluorogenic substrate followed by fluorimetric quantification of the product formed and released into incubation medium. For each individual P450 activity, different fluorescence probes were examined, and the one showing the best properties (highest metabolic rates, lowest background fluorescence) was selected: 3-cyano-7-ethoxycoumarin for CYP1A2 and CYP2C19, coumarin for CYP2A6, 7-ethoxy-4-trifluoromethylcoumarin for CYP2B6, dibenzylfluorescein for CYP2C8, 7-methoxy-4-trifluoromethylcoumarin (MFC) for CYP2C9 and CYP2E1, 3-[2-(N,N-diethyl-N-methylammonium)ethyl]-7-methoxy-4-methylcoumarin for CYP2D6, and 7-benzyloxy-4-trifluoromethylcoumarin for CYP3A4. Fluorescence-based assays are highly sensitive and allow the simultaneous measurement of a large number of samples using plate readers, thus enhancing sample throughput. Major advantages over high-throughput assays in subcellular fractions are that, as living cells are used, manual handling and enzyme damage are minimized, the endoplasmic reticulum of the cells remains intact, exogenous cofactors or NADPH-regenerating systems are not required, and transport processes are maintained. These assays can be applied to preliminary screening of inhibitory effects of new drugs on individual P450 enzymes. After comparison of the results obtained using the fluorescent probes in intact P450-expressing cells and those obtained using the high-performance liquid chromatography-based selective assays in the same cells, in primary human hepatocytes or in human liver microsomes, a fairly good agreement was found.