In a previous study, we investigated physical methods to reduce whole-body, diet-related autofluorescence interference in several mouse strains through changes in animal diet. Measurements of mice with an in vivo multispectral imaging system over a 21-day period allowed for the quantification of concentration changes in multiple in vivo fluorophores. To be an effective instrument, a multispectral imaging system requires a priori spectral knowledge, the form and importance of which is not necessarily intuitive, particularly when noninvasive in vivo longitudinal imaging studies are performed. Using an optimized spectral library from a previous autofluorescence-reduction study as a model, we investigated two additional spectral definition techniques to illustrate the results of poor spectral definition in a longitudinal fluorescence imaging study. Here we systematically evaluate these results and show how poor spectral definition can lead to physiologically irrelevant results. This study concludes that the proper selection of robust spectra corresponding to each specific fluorescent molecular label of interest is of integral importance to enable effective use of multispectral imaging techniques in longitudinal fluorescence studies.