Infrared spectroscopy has emerged as a promising technique for studying the composition of biological samples like lipids that play important roles in cellular functions and are involved in various diseases. For this reason, lipids are a target of interest in many biomedical studies. The objective of the present study is to utilize Fourier-Transform Infrared (FT-IR) spectroscopy to examine the main lipid components of human cells (phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, C18 ceramide, sphingosine-1-phosphate, ceramide-1-phosphate, sphingomyelin, cholesterol, and triolein). FT-IR analysis on the previously mentioned lipid samples was performed in Attenuated Total Reflection (ATR) mode. The obtained spectra clearly evidence the contributions of the different functional groups that are present in the examined samples. Detailed assignments of spectral features were carried out in agreement with the literature. Similarities and differences among the different types of commercial lipid samples are evidenced and discussed, with particular attention to phospholipid and sphingolipid components. A quantitative analysis of phosphatidylinositol and sphingomyelin spectra using a ratiometric approach is reported. Moreover, a reconstruction procedure of FT-IR spectra of complex lipids useful for chemometrics applications is described. These representative examples of the potential use of the results of the present study can certainly contribute to a larger use of FT-IR spectroscopy in lipidomics.