Fluorescence measurements can provide information about changes in the biochemical, functional and structural characteristics of fluorescent bio-molecular complexes in tissues and cells (e.g. structural proteins, enzyme metabolic co-factors, lipid components, and porphyrins). Typically, these changes are a result of either pathological transformations or therapeutic interventions. We research the development of instrumentation that utilizes label-free fluorescence lifetime contrast to detect and evaluate these molecular changes in vivo in patients and methodologies conducive to near-real time diagnosis of tissue pathologies. This presentation overviews clinically-compatible multispectral fluorescence lifetime imaging techniques developed in our laboratory and their ability to operate as stand-alone diagnostic devices, integrated in a biopsy needle and in conjunction with the da Vinci surgical robot. We present pre-clinical and clinical studies in patients that demonstrate the potential of these techniques for intraoperative assessment of brain tumors and head and neck cancer and intravascular evaluation of atherosclerotic cardiovascular disease. Current results demonstrate that intrinsic fluorescence signals can provide useful contrast for delineation distinct types of tissues including tumors intraoperatively. Challenges and solutions in the clinical implementation of these techniques are discussed.