Most of the methods dedicated to the monitoring of metabolic responses from isolated mitochondria are based on whole-population analyses. They rarely offer an individual resolution though fluorescence microscopy allows it, as demonstrated by numerous studies on single mitochondria activities in cells. Herein, we report on the preparation and use of microwell arrays for the entrapment and fluorescence microscopy of single isolated mitochondria. Highly dense arrays of 3 μm mean diameter wells were obtained by the chemical etching of optical fiber bundles (850 μm whole diameter). They were manipulated by a micro-positioner and placed in a chamber made of a biocompatible elastomer (polydimethylsiloxane or PDMS) and a glass coverslip, on the platform of an inverted microscope. The stable entrapment of individual mitochondria (extracted from Saccharomyces cerevisiae yeast strains, inter alia, expressing a green fluorescent protein) within the microwells was obtained by pretreating the optical bundles with an oxygen plasma and dipping the hydrophilic surface of the array in a concentrated solution of mitochondria. Based on the measurement of variations of the intrinsic NADH fluorescence of each mitochondrion in the array, their metabolic status was analyzed at different energetic respiratory stages: under resting state, following the addition of an energetic substrate to stimulate respiration (ethanol herein) and the addition of a respiratory inhibitor (antimycin A). Statistical analyses of mean variations of mitochondrial NADH in the population were subsequently achieved with a single organelle resolution.