Triplet imaging is a novel optical technique that allows investigating oxy- gen metabolism at the single cell and the sub-cellular level. The method combines high temporal and spatial resolution, which are required for the monitoring of fast kinetics of oxygen concentration in living cells. Calibration and validation is demon- strated with a titration experiment using L-Ascorbic Acid with the enzyme Ascor- base oxidase. The method was applied to a biological cell system, employing as reporter a cytosolic fusion protein of β-galactosidase with SNAP-tag labeled with tetramethylrhodamine. Oxygen consumption in single smooth muscle cells A7r5 during an [Arg8]-vasopressin-induced contraction is measured. The results indicate a consumption leading to an intracellular oxygen concentration that decays mono- exponentially with time. This is in good agreement with previously reported mea- surements of oxygen consumption in skeletal muscle fibers.