Understanding the role of lipids in protein function is key to obtaining details about cellular signaling pathways. Among the various methods available, label-free differential scanning fluorimetry has several advantages. This technique uses the intrinsic fluorescence of tryptophan and tyrosine residues, requires minute sample amounts, enables rapid, high-throughput analysis, and allows direct comparison of protein-lipid interactions in lipid bilayers and with water-soluble lipid analogues. Here, we present a detailed protocol for monitoring the interaction between human full-length Akt1 and phosphatidylinositol-3,4,5-trisphosphate (PIP₃), either in solution with short-chain PIP₃ (4:0/4:0) or within membranes containing PIP₃ (16:0/16:0). This versatile protocol can easily be adapted to study other protein-ligand interactions.