The ability to bind to and translocate across lipid bilayers is of paramount importance for the extracellular administration of intracellularly active compounds in cell biology, medicinal chemistry, and drug development. A combination of the so-called uptake and release experiments performed by high-sensitivity isothermal titration calorimetry provides a powerful and universally applicable tool for measuring membrane binding and translocation of various compound classes in a label-free manner in solution. The protocol presented here is designed for a quantitative analysis of microcalorimetric uptake and release titrations. In contrast with simpler approaches described previously, it is applicable also to electrically charged solutes, such as peptides and proteins, experimentally and clinically relevant surfactants, drugs, metal ions, and other ionic compounds.