Non-alcoholic fatty liver disease (NAFLD) is a common liver lesion that is untreatable with medications. Glucagon-like peptide-1 receptor (GLP-1R) agonists have recently emerged as a potential NAFLD pharmacotherapy. However, the molecular mechanisms underlying these drugs' beneficial effects are not fully understood. Using Fourier transform infrared (FTIR) spectroscopy, we sought to investigate the biochemical changes in a steatosis cell model treated or not with the GLP-1R agonist Exendin-4 (Ex-4). HepG2 cells were made steatotic with 400 µM of oleic acid and then treated with 200 nM Ex-4 in order to reduce lipid accumulation. We quantified steatosis using the Oil Red O staining method. We investigated the biochemical alterations induced by steatosis and Ex-4 treatment using Fourier transform infrared (FTIR) spectroscopy and chemometric analyses. Analysis of the Oil Red O staining showed that Ex-4 significantly reduces steatosis. This reduction was confirmed by FTIR analysis, as the phospholipid band (C=O) at 1740 cm in Ex-4 treated cells is significantly decreased compared to steatotic cells. The principal component analysis score plots for both the lipid and protein regions showed that the untreated and Ex-4-treated samples, while still separated, are clustered close to each other, far from the steatotic cells. The biochemical and structural changes induced by OA-induced lipotoxicity are at least partially reversed upon Ex-4 treatment. FTIR and chemometric analyses revealed that Ex-4 significantly reduces OA-induced lipid accumulation, and Ex-4 also restored the lipid and protein biochemical alterations caused by lipotoxicity-induced oxidative stress. In combination with chemometric analyses, FTIR spectroscopy may offer new approaches for investigating the mechanisms underpinning NAFLD.