Fluorescence spectroscopy is an effective and versatile tool to analyze substances. The basis of the analysis is the light scattering and fluorescence principles. Analyzing optimal wavelengths to achieve the greatest signal of fluorescence while reducing any amplification of the signal from scattered light is an important skill needed in modern substance analysis. This work focuses on implementing and refining the skills required to optimally perform spectrofluorometry on the substance dichlorofluorescein, that spectrofluorometry is a viable method to determine the concentration of an unknown and that fine-tuning instrument settings to a substance yields optimal results.