A laboratory application with a strong component in analytical chemistry was designed for undergraduate students, in order to introduce a current problem in the environmental science field, the water contamination by antibiotics. Therefore, a simple and rapid method based on direct injection and high performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed and optimized for the determination of amoxicillin in river water. Students learned the main optimization steps for the improvement of the LC-MS/MS methodology and the correct procedure to validate an analytical method (draw a calibration curve, determine detection and quantification limits, precision, and accuracy). This laboratory experiment was successfully applied by students and enables the analysis of a large number of samples in a short period of time because of the short run time (3 min).