High-performance methods of testing of drug candidates for properties of pharmacokinetics and pharmacodynamics importance, in particular lipophilicity and acidity, are necessary to overcome innovation stagnation in the pharmaceutical industry. Reversed-phase high-performance liquid chromatography (RP HPLC) might be a unique tool for the determination of both pKa and the apparent (pH-dependent) partition coefficient, applicable in high-throughput analysis of multicomponent mixtures, e.g., samples originating from automated synthesis. In this work, the pH/organic modifier gradient RP HPLC is presented as a means of simultaneous determination of an analyte's acidity and lipophilicity. The approach consists of retention measurements in a series of methanol gradient runs differing in pH range and duration of the gradient. Two different models of the influence of pH on retention in organic modifier gradient RP HPLC are compared regarding the quality of the simultaneously determined lipophilicity and dissociation constants. Advantages of the proposed approach over currently employed procedures are that it can be applied to compound mixtures, it requires only minute amounts of substances, and pKa values can be determined in the range 3-10 units and lipophilicity in the range 0-7 units. Verification of the reliability of the parameters determined by the new method was demonstrated on a series of 93 acidic and basic drug analytes.