Fluorescence lifetime imaging (FLIm) is an optical spectroscopic imaging technique capable of real‐time assessments of tissue properties in clinical settings. Label‐free FLIm is sensitive to changes in tissue structure and biochemistry resulting from pathological conditions, thus providing optical contrast to identify and monitor the progression of disease. Technical and methodological advances over the last two decades have enabled the development of FLIm instrumentation for real‐time, in situ, mesoscopic imaging compatible with standard clinical workflows. Herein, we review the fundamental working principles of mesoscopic FLIm, discuss the technical characteristics of current clinical FLIm instrumentation, highlight the most commonly used analytical methods to interpret fluorescence lifetime data and discuss the recent applications of FLIm in surgical oncology and cardiovascular diagnostics. Finally, we conclude with an outlook on the future directions of clinical FLIm. Mesoscopic fluorescence lifetime imaging (FLIm) is an optical spectroscopic imaging technique for real‐time assessments of tissue properties in clinical settings, used to monitor disease. This report describes the fundamental principles of FLIm, discusses the technical characteristics of current clinical FLIm instrumentation, highlights commonly used methods to analyze and interpret FLIm data and summarizes recent applications in surgical oncology and cardiovascular disease. It concludes with an outlook to the future of clinical FLIm.