Recently, it has been reported that anti-viral drugs, such as indinavir and lopinavir (originally targeted for HIV), also inhibit E6-mediated proteasomal degradation of mutant p53 in E6-transfected C33A cells. In order to understand more about the mode-of-action(s) of these drugs the metabolome of HPV16 E6 expressing cervical carcinoma cell lines was investigated using mass spectrometry (MS)-based metabolic profiling. The metabolite profiling of C33A parent and E6-transfected cells exposed to these two anti-viral drugs was performed by ultra performance liquid chromatography (UPLC)-MS and gas chromatography (GC)-time of flight (TOF)-MS. Using a combination of univariate and multivariate analyses, these metabolic profiles were investigated for analytical and biological reproducibility and to discover key metabolite differences elicited during anti-viral drug challenge. This approach revealed both distinct and common effects of these two drugs on the metabolome of two different cell lines. Finally, intracellular drug levels were quantified, which suggested in the case of lopinavir that increased activity of membrane transporters may contribute to the drug sensitivity of HPV infected cells.