Inhibition of protein deacetylation enzymes, alone or in combination with standard chemotherapies, is an exciting addition to cancer therapy. We have investigated the effect of deacetylase inhibition on the metabolism of glioblastoma cells. 1H NMR metabolomics analysis was used to determine the major metabolic changes following treatment of two distinct glioblastoma cell lines, U373 and LN229, with five different histone deacetylase (HDAC) inhibitors, as well as one inhibitor of NAD+-dependent protein deacetylases (SIRT). The addition of the standard glioblastoma chemotherapy agent, temozolomide, to the HDAC and SIRT treatments led to a reduction in cell survival, suggesting a possibility for combined treatment. This study shows that distinct glioblastoma cell lines, with different metabolic profiles and gene expression, experience dissimilar changes following treatment with protein deacetylase inhibitors. The observed effects of inhibitors on mitochondrial metabolism, glycolysis and fatty acid synthesis suggest possible roles of protein deacetylases in metabolism regulation. Metabolic markers of the effectiveness of anti-protein deacetylase treatments have been explored. In addition to known deacetylation inhibitors, three novel inhibitors have been introduced and tested. Finally, 1H NMR analysis of cellular metabolism is shown to be a fast, inexpensive method for testing drug effects.