A novel bioluminescence assay has been developed for measuring pyruvate within sections of snap-frozen tissue in a quantitative manner as well as with a spatial resolution on a microscopical level. The assay was verified via HPLC and two independent photometric tests. The novel assay makes it possible to determine pyruvate concentrations in cryosections in the range of 0–5.0 μmol/g tissue (dry weight). Based on the analysis of samples of given pyruvate concentrations, the assay exhibits a recovery with a deviation ≤15%. The minimal detectable amount was 0.02 pmol based on a 20 μm thick tissue section with an area of 1 cm2. Combination of the already established imaging bioluminescence techniques for ATP, glucose, and lactate with the novel pyruvate assay allows for a comprehensive characterization of the metabolic profile of individual tumors. As the redox state of cancer cells can be critical for the efficiency of irradiation and a number of chemotherapeutics, and as pyruvate and lactate are known to have radical scavenger functions, we hypothesize that the novel bioluminescence assay may be used for measuring the pretherapeutic lactate-to-pyruvate ratio which may predict the radiosensitivity of individual malignancies.