Multiphoton laser scanning microscopy (MPLSM) utilizing techniques such as multiphoton excitation (MPE), second harmonic generation (SHG), and multiphoton fluorescence lifetime imaging and spectral lifetime imaging (FLIM and SLIM, respectively) are greatly expanding the degree of information obtainable with optical imaging in biomedical research. The application of these nonlinear optical approaches to the study of breast cancer holds particular promise. These noninvasive, multidimensional techniques are well suited to image exogenous fluorophores that allow relevant questions regarding protein localization and signaling to be addressed both in vivo and in vitro. Furthermore, MPLSM imaging of endogenous signals from collagen and fluorophores such as nicotinamide adenine dinucleotide (NADH) or flavin adenine dinucleotide (FAD), address important questions regarding the tumor-stromal interaction and the physiologic state of the cell. We demonstrate the utility of multimodal MPE/SHG/FLIM for imaging both exogenous and/or endogenous fluorophores in mammary tumors or relevant 3-D systems. Using SLIM, we present a method for imaging and differentiating signals from multiple fluorophores that can have overlapping spectra via SLIM Plotter—a computational tool for visualizing and analyzing large spectral-lifetime data sets.