Virotherapy, enabled by recent advances in the transdisciplinary field of biotechnology, has emerged as a powerful tool for use in anticancer treatment, gene therapy, immunotherapy, etc. Examining the effects of viruses and virus-derived immune-modulating therapeutics is of great fundamental and clinical interest. Here we describe a sample preparation protocol for metabolite extraction from virus-infected tissue, in addition to liquid chromatography-mass spectrometry conditions essential for subsequent analysis. This metabolomics approach delivers highly sensitive and specific metabolite information on various biospecimens. Such an approach may be adopted to monitor biological changes in over 30 relevant metabolic pathways in response to viral infection and also viral therapeutics.