Evolution has provided life on earth with several methods of generating cellular energy. None is more complex than the metabolism of mammalian cells. Fluorescence lifetime imaging [1-2] is a sensitive technique to investigate the co-enzymes NAD(P)H (reduced nicotinamide adenosine dinucleotide (phosphate)[H]) and FAD (oxidized flavine adenine dinucleotide), the former being present in cytosolic glycolysis and mitochondrial citric acid and ETC/OXPHOS, the latter mainly in the mitochondria. Both co-enzymes are auto-fluorescent in their reduced (NAD(P)H) and oxidized (FAD) forms and therefore suitable targets for light microscopy exploration . Disturbed and/or remodeled cellular metabolism is present in a number of human pathologies, e.g. cancer, neurodegenerative diseases, diabetes, inflammatory processes, to name a few, and monitoring these changes provides critical information for diagnosis and disease progression . Tryptophan (Trp), an essential amino acid and also auto-fluorescent, is used as a measure of cellular protein abundance and has been linked to cancer investigations 5-6]. The characterization of these endogenous molecules help us to understand the heterogeneous distribution of the metabolic signals or mapping of the metabolic signals in cancer cells and tissues. The traditional intensity-based redox ratio includes intensity artefacts due to differential absorption and scattering in tissues and usage of various average excitation intensity levels at different depths. The developed FLIM assay, fluorescence lifetime redox ratio (FLIRR), based on discrete ROIs (2x2 pixels), which mirror intensity-based morphology and measures the heterogeneous environment of the lifetime distribution in the prostate cancer cells . Glucose uptake and glycolysis proceeds about ten times faster in cancer than in non-cancerous cells or tissues. Therefore, we assessed the glycolytic activity in prostate cancer in comparison to normal cells upon glucose stimulation by analyzing the NADH and Trp lifetime distribution and efficiency of energy transfer (E%). The magnitude of the NADH a2% and E% distribution was higher in prostate cancer cells as compared to the normal cells. Hence, the FLIRR method and Trp lifetimes can be used as a biomarker to understand metabolic activity in prostate cancer and upon chemotherapeutic interventions.