Langmuir films are monolayers of water-insoluble molecules formed on aqueous subphases due to the forces of self-assembly. The increased interest in Langmuir monolayers is primarily related to their use as models of biological membranes. The possibility of studying the incorporation and behavior of bioactive compounds (i.e., drugs) in the mem0brane structure seems to be particularly important. The Langmuir technique enables the investigation of such aspects as orientation of molecules at the interface and interactions with lipids present in membranes. Here, we present the research methodology on an example of a chain-oxidized cholesterol derivative using Langmuir monolayers: from single-component to mixed films. The protocol also describes the application of related complementary in situ techniques, such as Brewster angle microscopy (BAM; film texture imaging), electric surface potential change (ΔV measurements), and polarization modulation-infrared reflection-absorption spectroscopy (PM-IRRAS; IR spectra).