The LEDIF (LED-induced fluorescence) is an in situ optical instrument that utilizes fluorescence, absorbance, and scattering to identify and quantify substances in water bodies. In this study, matrix effects on fluorescence signals caused by inner filtering, temperature, intramolecular deactivation, turbidity, and pH were investigated, and compensation equations developed to correct measured values and improve accuracy. Multiple simultaneous matrix effect corrections were demonstrated with a laboratory sample subjected to known interferences and physical conditions. In general, compensation was found to be important to improve the accuracy of fluorescence measurements.