A technique for simultaneously measuring changes in extracellular glucose, lactate, and oxygen concentrations in conjunction with acidification rates on a Cytosensor Microphysiometer is described. Platinum electrodes are inserted into the standard Cytosensor plunger head and modified with enzymes and biocompatible polymeric films. The lactate and glucose oxidase enzymes catalyze the reaction of lactate and glucose. An end product of these catalyses, H2O2, is measured amperometrically. Extracellular oxygen is also measured amperometrically, while the acidification rate is measured potentiometrically by the Cytosensor. Useful information is obtained during the Cytosensor stop-flow cycles, which produce increasing or decreasing peaks, owing to the production of lactic and carbonic acid and consumption of glucose and oxygen by the cells. Fabrication of the modified sensor head and deposition of the electrode films is detailed, and the operation of the technique is described and illustrated by the simultaneous measurement of all four analytes during the addition of 20 mM fluoride to mouse fibro blast cells.