PQ depletion and metabolite production from hepatocyte incubation monitored via UHPLC-MS/MS demonstrated a faster metabolic rate for (−)-PQ as compared to (+)-PQ. The rate of metabolism was profiled against time, yielding an initial half-life of 3.65 and 1.08 h for (+)- and (−)-PQ, and elimination rate constants (ʎ) of 0.19 and 0.64 h−1, respectively. The calculated in vitro intrinsic clearance (Clint) was 2.55 and 8.49 (µL/min)/million cells for (+) and (−)-PQ, respectively. Metabolism was observed via oxidative deamination of the terminal amine, hydroxylation of the quinoline ring, and what appears to be a third path, carbamoylation and glucuronidation of the terminal amine (m/z 480). The major deaminated metabolites were CPQ, PQ terminal alcohol, a cyclized side chain derivative from the aldehyde (m/z 241), cyclized carboxylic acid derivative (m/z 257), a quinone-imine product of hydroxylated CPQ (m/z 289), CPQ glucuronide (m/z 451), and the glucuronide of PQ alcohol (m/z 437). All were preferentially generated from the (-)-PQ. The major quinoline oxidation product (m/z 274) and 4-hydroxyprimaquine were preferentially formed from (+)-PQ. A prominent conjugate (m/z 422) (seemingly a glycosylated PQ, (but perhaps reflecting a fragment or degradation product of another metabolite) was also preferentially (+)-PQ-derived. The PQ carbamoyl glucuronide metabolite (m/z 480) accumulated linearly over the incubation period, and was exclusively generated from (+)-PQ.