Eukaryotic cells use a series of membrane transporters to control the movement of lipids across their plasma membrane. Several tools and techniques have been developed to analyze the activity of these transporters in the plasma membrane of mammalian cells. Among them, assays based on fluorescence microscopy in combination with fluorescent lipid probes are particularly suitable, allowing visualization of lipid internalization in living cells. Here, we provide a step-by-step protocol for mammalian cell culture, lipid probe preparation, cell labeling, and confocal imaging to monitor lipid internalization by lipid flippases at the plasma membrane based on lipid probes carrying a fluorophore at a short-chain fatty acid. The protocol allows studying a wide range of mammalian cell lines, to test the impact of gene knockouts on lipid internalization at the plasma membrane and changes in lipid uptake during cell differentiation.