Amphiphilic monolayers formed at the soft air/liquid interface are easy-to-handle and versatile model systems for material and life sciences. Helmuth Möhwald was one of the pioneers in this field. Over the last few decades, total-reflection X-ray fluorescence (TRXF) has become an important analytical tool for the investigation of monolayer interactions with ions. Here, the theoretical background of TRXF is described, and practical aspects are discussed. The experimentally determined fluorescence intensity from the adsorbed ions can be interpreted quantitatively either by a calibration procedure utilizing monolayers with known charge density or by calibration with respect to the bare aqueous surface. Both calibration approaches yield quantitatively consistent results within <10% accuracy. Some examples demonstrating the power of TRXF for the study of ion adsorption to charged and noncharged monolayers as well as for the characterization of the physicochemical properties of novel cationic lipids used for improved gene delivery are given.