BACKGROUND: Metabolic dysfunction-associated steatotic liver disease is defined by hepatic lipid overload resulting in a metabolic shift and subsequent mitochondrial impairment. Diagnosis currently relies on tissue biopsy and non-invasive tests. However, these have drawbacks, including subjective histology scoring and relatively low sensitivity, highlighting the need for more robust and reproducible methodologies. METHODS: Fluorescence lifetime imaging microscopy visualises the metabolic state of cells by measuring the autofluorescence lifetime of metabolites, effectively avoiding the need for exogenous labelling. This technique was applied to a broad range of models, spanning from a hepatocyte cell line to a human tissue slice model, to investigate metabolic changes across disease conditions. RESULTS: Here, by utilising the metabolic dysfunction associated with steatotic liver disease, we propose a time-efficient method and introduce an index as a quantitative output to assess the metabolic state of human liver biopsies. The index encapsulates features of metabolic dysfunction that directly report on the disease state. These findings using lifetime imaging are substantiated by extensive analysis of structural and functional mitochondrial dysfunction. CONCLUSIONS: Measuring fluorescence lifetime can capture features of metabolic change that standard histological methods do not. Correlating the results to established techniques of histological evaluation highlights the potential of this method to enhance characterisation and speed of biopsy results in metabolically implicated diseases.