Resistance to clinically useful therapeutic antibiotics is an ever-increasing phenomenon seen in a range of bacterial species including those pathogenic to man. There are diverse mechanisms which contribute to inherent and acquired resistance to antibiotics. Gram-negative bacteria are commonly intrinsically more resistant to many drugs as a result of their cell structure and the activity of multidrug efflux pumps. Measurement of the accumulation of antibiotics and the contribution of active efflux has proved important in understanding the mechanisms of resistance to many antibiotics and how bacteria can become multidrug-resistant. Multidrug efflux pumps often have broad substrate ranges allowing detection of their activity by measurement of the accumulation of antibiotic substrates or a range of fluorescent substrates, which can be easily used as markers of efflux activity. This chapter describes methods for the detection of efflux pump activity on Gram-negative bacteria.