This study focuses on the spectrofluorimetric behavior of the camptothecin derivative 7-ethyl-10-hydroxycamptothecin (SN-38) alone and in the presence of organized media and also on its potential analytical applications. SN-38 displays native fluorescence in both lactone and carboxylate form, which has been the base for development of two spectrofluorimetric methods, one for the lactone form (acidic media) and another for the carboxylate form (basic media). In an attempt to improve the understanding of SN-38, its interaction with several cyclodextrins and surfactants has been studied using spectrofluorimetry. Consequently, the optimal working conditions for the determination of SN-38 have been established in both the presence and the absence of organized media. The proposed methods were applied to human urine, using liquid–liquid extraction for clean-up of the samples, with satisfactory recoveries. No interference of the urine matrix was observed.