N-in-1 (or cassette) dosing pharmacokinetics (PK) has been used in drug discovery for rapid assessment of PK properties of new chemical entities. However, because of potential for drug-drug interactions this procedure is still controversial. This study was to retrospectively evaluate the N-in-1 dosing approach in drug discovery with an emphasis on the potential for drug-drug interactions. The systemic clearance, volume of distribution, oral bioavailability, and renal excretion of the 31 lead compounds in rats, dogs or chimpanzees were significantly correlated between the N-in-1 dosing and discrete studies with r values of 0.69, 0.91, 0.53, and 0.83 (p < 0.005 for all), respectively. PK parameters for 11 quality control compounds which were involved in 194 N-in-1 studies for screening approximately 1000 compounds had coefficient of variations of less than 70%. The intrinsic microsomal clearances generated from the N-in-1 and discrete incubations were nearly identical (r = 0.97, p < 0.0001). The intrinsic clearances of quality control compound from the N-in-1 incubations were consistent with its discrete CL(int) estimate (cv: 5.4%). Therefore, N-in-1 dosing is a useful approach in drug discovery to quickly obtain initial PK estimates. Potential drug-drug interactions that result in confounding PK estimates do not occur as frequently as expected.