Due to its fundamental role in cellular energetics, metabolic regulation and cellular signalling, determination of intracellular adenosine-5'-triphosphate (ATP) is an irreplaceable tool in the characterization of cellular physiology. Classical protocols describing the measurement of ATP from cells usually suffer from the use of relatively high reaction volumes and the according use of cuvettes for ATP determination, thus making them impractical for processing high sample numbers. We therefore present two methods for the fast and reliable measurement of intracellular ATP levels of cells cultured in 96-well microplates. The procedures are compared with each other concerning detection limit, dynamic range, precision and practicability. Both methods described allow the fast and standardized measurement of intracellular ATP using the 96-well microplate format that is applicable for both, high-throughput cell culture and subsequent determination of intracellular ATP.