Pyruvate kinase is a key enzyme in glycolytic pathway that catalyzes the transphosphorylation between phosphoenolpyruvate and ADP to yield ATP and Pyruvate. Geobacillus stearothermophillus has a stable pyruvate kinase with determined crystal structure that composed of four separate domains. Given that limited proteolysis experiments can be successfully used to probe conformational features of proteins, in this study we obtained useful information on Geobacillus pyruvate kinase using limited proteolysis with two proteases that have different substrate specificity and optimum temperature of activity, trypsin and thermolysin. Proteolytic patterns at different temperatures indicate that resulting fragments were the same but the rate of digestion increased with temperature. In the next step, Sucrose and Glycine were used to examine the effects of additives on stability and activity of pyruvate kinase. Limited proteolysis was carried out at 37 °C by trypsin and at 30, 55 and 60 °C in presence of thermolysin, in the absence and presence of different concentrations of sucrose (0– 1.5 M) and glycine (0–1.5 M). We observed that stabilization of pyruvate kinase by this osmolytes is concentration dependent and the rate of limited proteolysis in presence of additives, at temperatures above 60 °C decrease; however, there was no any effect on proteolytic patterns. In all experiments the activity of pyruvate kinase was determined with a couple assay methods by luciferase. A clear correlation was observed between proteolytic digestion and enzyme activity. This study reveals a number of flexible and protease-prone regions of pyruvate kinase that exist regardless of the environmental conditions.