Poor therapeutic outcomes have been attributed to inter-individual and interethnic variability in cytochrome P450 (CYP450)-dependent metabolism and altered drug retention associated with P-glycoprotein (P-gp). An individualized pharmacotherapeutic approach would benefit the genetically diverse South African population. The study aim was to develop a validated, targeted, analytical method to quantify seven probe drugs and their metabolites in dried blood spots (DBS) using the Mitra™ volumetric absorptive micro-sampling device for blood collection. An Agilent liquid chromatography (LC) system coupled to a Sciex 4000 QTRAP tandem mass spectrometer (MS) was used for method optimization and validation. Targeted LC-MS/MS methods were validated according to ICH guidelines. The validated LC-MS/MS method met the required bioanalytical standards for specificity, sensitivity, linearity, accuracy, precision, carry-over, and stability. This study successfully validated the use of DBS, collected with the Mitra™ microsampling device, to measure expected probe drug and metabolite concentrations using the “Geneva phenotyping cocktail” for the purpose of simultaneous phenotyping of in vivo CYP450 metabolic activity of the CYP1A2, -2B6, -2C9, -2C19, -2D6, and -3A4 enzymes, and P-gp transport activity. However, statistical analysis using Bland-Altman plots showed that not all analytes exhibited linear distribution pharmacokinetics between DBS and plasma, which influenced the predicted vs real plasma concentrations based on measurements in a DBS matrix. These findings were attributed to the blood-to-plasma concentration ratio, the physicochemical properties and stability of the analytes, and the extraction efficiency of the Mitra™ sampler. More research is required before DBS sampling can be substituted for conventional plasma sampling