Steady-state fluorescence spectroscopy (SSFS) is frequently used to provide thermodynamic parameters of intermolecular interactions that occur close to fluorophore sites. To use this technique, at least one of the interacting molecules must be capable of emitting fluorescence after absorbing electromagnetic energy. SSFS is a relatively simple technique, however, it is very important to know some properties of the analyzed molecules, such as the wavelength of maximum absorption of electromagnetic energy and the solubility in the working solvent. Knowing these characteristics helps to fix errors in analytical measurements, such as the inner-filter effect, for example. Here, we describe the step-by-step protocol for carrying out the practical experiment and some precautions that need to be taken to obtain reliable analytical data in intermolecular interaction researches using the spectrofluorometer. We will also discuss some mathematical models used in data analysis to obtain the thermodynamic parameters of interaction.