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Lipolysis is an important biological process responsible for delivering fatty acids appropriately to meet energy needs. An unbalanced amount of free fatty acids is associated with insulin resistance and an increase in triglyceride levels. This process is related to the degradation of triglycerides in differentiated adipocytes. Typically, the rate of lipolysis in adipocytes is measured using commercial kits (for triglyceride quantification or glycerol release), or by assessing the mRNA levels of lipases and regulatory proteins, and in some cases, by chromatographic techniques to quantify free fatty acids. Both techniques are time-consuming and expensive, which can be a disadvantage in the search for new compounds to reduce fat accumulation in adipose tissue. Therefore, Fourier transform infrared spectroscopy (FTIR) can be a valuable tool for measuring lipolysis due to its ease of operation, rapid analysis time, and low cost.