Fluorescence imaging is an emerging imaging technique that has shown many benefits for clinical care. Currently, the field is in rapid clinical translation, and an unprecedented number of clinical trials are performed. Clinicians are inundated with numerous opportunities and combinations of different imaging modalities. To streamline this process, a multidisciplinary approach is needed with drug discovery, software and systems engineering, and translational medicine. Here, we discuss the main constituents of a uniform fluorescence imaging protocol to match the clinical need and ensure consistent study designs and reliable data collection in clinical trials. In an era in which the potential of fluorescence imaging has become evident, consistent conduct of studies, data analysis, and data interpretation is essential for implementation into the standard of care.