Purpose: The present study is a compilation of methodologies developed in order to evaluate the effects of addition of a fluorescent agent, rhodamine B, to resin-based materials. The intent of the work was to develop a systematic methodology that accounts for variables not shown to be of concern in past testing, but may significantly affect interpretation of the resulting images and material properties. Materials and Methods: Different methodologies were specifically developed to evaluate factors affecting the use of the fluorescent agent rhodamine B in the identification of resin-based materials. The influence of dye concentration was evaluated with respect to adhesive polymerization reaction kinetics and bond strength of the resin into which the dye is added. The pH of the dye-solvent solution was also considered. Additionally, the mass and percentage of rhodamine B leached into different media over time was determined. Results: It was demonstrated that a specific dye must be used with caution, and the concentration of a fluorescent agent is important with respect to both the conversion and bond strength of the resin to which the dye is added. An alcoholbased dye solution also produced an extremely high amount of dye leaching from polymerized specimens. The adhesive pH was not altered. Conclusion: The standardized methodology developed here to analyze restoration interfaces using a fluorescent dye can lessen the impact of variables when interpreting results. Keywords: fluorescence microscopy, fluorescent dyes, dental adhesives