Multispectral fluorescence imaging (MSFI) is a rapidly growing field with broad applications in both preclinical and clinical settings. Application of this novel technology in small-animal imaging and microscopy produces enhanced sensitivity and reliable quantification and resolves multiple simultaneous signals. MSFI flow cytometry can quantify multiple fluorescent parameters with morphologic or subcellular spatial details on millions of cells. MSFI has the potential to improve the accuracy of disease detection or differentiation and intrasurgical metastatic diagnosis, guide neurosurgeries, and monitor treatment response.