Human endothelial cells (ECs) have been employed to monitor the protein changes induced by [3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one] (3PO), a compound able to inhibit the glycolytic flux partially and transiently and to reduce pathological angiogenesis in a variety of disease models. Normal and TNFα induced inflamed ECs were incubated with and without 3PO at a concentration (20 μM) able to inhibit cell proliferation without cell death. At the end of the incubation period, samples were submitted to the following steps: (a) whole protein extraction, reduction, alkylation, and digestion by trypsin; (b) peptide separation by nano-LC-MS/MS analysis using a high-resolution mass spectrometer; (c) data analysis including protein identification, quantification, and statistical analysis. An altered protein expression profiling in combination with protein network analysis was employed by using a mass spectrometry-based label-free quantification approach to explore the underlying mechanisms of 3PO at cellular level.