AbstractSkin cancer is associated with abnormal cellular metabolism which if identified early introduces the possibility of intervention to prevent its progress to a deadly metastatic stage. This study combines multiphoton microscopy with fluorescence lifetime imaging (FLIM) using a syngeneic melanoma mouse model, to detect changes in metabolic state of single epidermal cells as a metabolic marker to monitor the progress of tumor growth. This method utilizes imaging of the ratio of the amounts of the free and protein‐bound forms of the intracellular autofluorescent metabolic co‐enzyme nicotinamide adenine dinucleotide (NADH). Here, we investigate the impact of the primary tumor lesion on the epidermal layers at three different growth stages of melanoma lesion compared to normal skin as a control. We showed a significant increase in the free‐to‐bound NADH ratio with the growth of the solid melanoma tumor, while concurrently the short and the long lifetime components of NADH remained constant. These results demonstrate the ability of FLIM for rapid, non‐invasive and sensitive assessment of melanoma progression revealing its potential as a diagnostic tool for melanoma detection and as an aid for melanoma staging.