Attenuated oncolytic viruses (OVs) are a promising alternative cancer therapy to mainstream methods such as radiotherapy and chemotherapy. OV therapy takes advantage of the defective antiviral response present in most cancer cells however heterogeneity amongst target cells and attenuation of OVs to increase their safety profiles has limited the efficacy of this treatment. We have developed novel small molecules named viral sensitizers (VSes) capable of enhancing viral infection and cancer-specific cell death. Liquid chromatography-mass spectrometry (LC-MS) methods were developed to study the pharmacokinetics (PK) and metabolomic activity of VSe1-28 via time course experiments. Parallel to this work, proteomic experiments were conducted to confirm the molecular target and mechanism of action of VSe1-28 through MRM and Auto-MS/MS methods. These new findings will aid in improving VSes and progress preclinical studies for clinical use in combination with OVs.