The objective of the validation of an analytical procedure is to demonstrate that it is suitable for its intended purpose (Rao 2018). Methods need to be validated or revalidated before their introduction into routine use (Agalloco 1995). The analytical validation performed in this research included linearity, percentage recovery, repeatability, matrix effect, the limit of detection, and the limit of quantification. The result of the linearity of the analytical method is presented in Table 3, and it was determined as described in the validation and linearity section (see Supplementary Information). The response area of the three analytes was linear to the measured concentrations of the standards. The R² values for the three compounds were 0.979, 0.998, 0.988, 0.989, and 0.983 for Nevirapine, Emtricitabine, Ritonavir, Efavirenz, and Tenofovir, respectively. The result showed that a good correlation was obtained between the peak areas and concentrations. The limit of detection (LOD) of an analytical technique is the lowest concentration of an analyte that can be detected or distinguished (though not quantified) from the noise of an analytical procedure or of an instrument. It is usually stated as a concentration at a signal-to-noise ratio of 3:1 (Bhardwaj et al. 2015; Vidushi et al. 2017). The limit of quantitation (LOQ), on the other hand, is the lowest concentration of an analyte that can be successfully quantified in a sample with satisfactory precision and accuracy under specified working conditions (Bhardwaj et al. 2015). It is usually calculated as a signal: noise ratio of 10:1 as recommended by ICH (Bhardwaj et al. 2015; Vidushi et al. 2017). Table 3 presents the LOD for the five pharmaceutical formulations and it varied from 0.12 to 0.16 ng/ml. LOQ ranged between 0.40 and 0.53 ng/ml. Triplicate measurements of peak areas of seven different concentrations (0.5–12 μg/L) for Nevirapine, Emtricitabine, Ritonavir, Efavirenz, and Tenofovir were determined using the LC-MS/MS. The plot of the area against concentration was used to prepare the seven-point calibration curves for each pharmaceutical compound as shown in Appendix A to Appendix E (see Supplementary information). Typical chromatograms for the calibration standard are presented in Appendix F to Appendix K (see Supplementary information).