In this study, the alkaline protease-production capacity of a Geobacillus subterraneus C2-1 isolate from the Çitgöl thermal spring was investigated and optimised using a Plackett-Burman experimental design. In addition to the incubation time, which was the most important parameter, other significant factors were the peptone, glucose, and MgSO4.7H2O concentrations, the activation time and the inoculum amount. The highest protease activity of the Geobacillus subterraneus C2-1 isolate was observed using 14.75 g L-1 glucose as the carbon source, 7.51 g L-1 yeast extract as the nitrogen source, an inoculum amount of 3.56%, a temperature of 56.35 °C, and a reaction time of 168 h. In the presence of 1.0 mM SDS, the protease activity did not change and even increased. Furthermore, 100 mM EDTA yielded a five-fold enhancement in specific activity. The presence of chemical denaturants, chelators, and even heavy metals did not alter the protease activity of the C2-1 isolate.