Cavities in proteins perform diverse functions such as substrate binding, enzyme catalysis, passage for transportation of small molecules, and protein oligomerization. Often, the physical properties of these cavities are closely linked to the protein function; such as the hydrophobic lipid-binding cavities in lipid-binding proteins (LBPs) that protect lipid substrates from the larger aqueous milieu. Therefore, the characterization of protein cavities can provide valuable insights into protein structure-function relationships, hinting toward their mechanism of action while aiding in the identification of ligand binding sites that are essential for drug discovery approaches. Several algorithms have historically been designed to identify and characterize the different types of cavities in protein structures. We summarize these algorithms and provide a step-by-step guide for locating and characterizing internal cavities in proteins using CICLOP by using ATP-binding cassette transporter A1 (ABCA1) as an example.