Attenuated viruses hold great potential in viral-based therapies including vaccine production and oncolytic virotherapy. Both applications are hindered by poor intercellular infection propagation due to genetic heterogeneity amongst target cells in regard to antiviral response functionality. Our collaborative research group has developed a novel solution to address this problem: first-in-class small molecules termed viral sensitizers (VSes) that potentiate attenuated viral infection in resistant cells. Liquid chromatography mass spectrometry (LC-MS) methods using various scanning modes were developed to characterize the poor physiochemical properties of the first lead compound. Furthermore, LC-MS methods were applied in a VSe library screen and quality assurance experiments. New lead compounds were identified with improved plasma stability and retained activity. Pharmacokinetic and metabolomic studies using lead VSes revealed that glutathione stability and tumour penetrance are important considerations for future experiments. MS-based and gel-based proteomic experiments used target identification techniques to elucidate the VSe mechanism of action.