In the crowded cellular milieu, biological processes require coordinated intermolecular interactions, conformational changes, and molecular transport that span a wide range of spatial and temporal scales. This complexity requires an integrated, noninvasive, multiscale experimental approach. Here, we develop a multimodal fluorescence microspectroscopy system, integrated on a single platform, to gain information about molecular interactions and their dynamics with high spatio-temporal resolution. To demonstrate the versatility of our experimental approach, we use rhodamine 123-labeled mitochondria in breast cancer cells (Hs578T), verified using differential interference contrast (DIC) and fluorescence (confocal and two-photon) microscopy, as a model system. We develop an assay to convert fluorescence intensity to actual concentrations in intact, individual living cells, which contrasts with conventional biochemical techniques that require cell lysates. In this assay, we employ two-photon fluorescence lifetime imaging microscopy (FLIM) to quantify the fluorescence quantum yield variations found within individual cells. Functionally driven changes in cell environment, molecular conformation, and rotational diffusion are investigated using fluorescence polarization anisotropy imaging. Moreover, we quantify translational diffusion and chemical kinetics of large molecular assemblies using fluorescence correlation spectroscopy. Our integrated approach can be applied to a wide range of molecular and cellular processes, such as receptor-mediated signaling and metabolic activation.