The acid dissociation constant (pK _a) is a key physicochemical parameter for characterizing active pharmaceutical ingredients (APIs). Early determination of pK _a values is highly desirable in drug discovery, pharmaceutical process research and formulation design. To overcome the challenges of limited sample availability and potential low purity of API samples at early stages of drug development, as well as to increase sample analysis throughput, a multiplexed 96-channel capillary electrophoresis with UV detection was evaluated as a practical approach for high throughput pK _a estimation of proprietary APIs in support of pharmaceutical research. Proprietary APIs with diverse structures were examined using the approach. The pK _a values were successfully determined with good accuracy and precision. System robustness was demonstrated and analysis of at least eight samples can be completed within 1 h. A rapid pK _a estimation procedure for marginally soluble APIs was proposed by performing single-point multiplexed CE–UV measurement without extrapolation using 10 or 20% methanol as co-solvent. Direct pK _a estimation of APIs using DMSO solution samples and crude reaction samples containing a large amount of solvents and reagents and high level of impurities was also demonstrated using the multiplexed CE–UV approach.