Extraction of proteins from extensively calcified osseous tissue, such as cortical bone has been particularly challenging for traditional methods of sample preparation. However, a comprehensive proteomic analysis of bone is only possible when the total protein constituency is effectively isolated. The efficiency of sample preparation is therefore a critical component of the analytical process. Historically, extraction of protein from bone required prolonged acid demineralization over several days to enable complete penetration of histochemical reagents to cellular components. Here we describe a method for the extraction of protein from ostrich tibia, which was used as a model sample to develop an extraction process that uses pressure cycling technology (PCT) and also which obviates the need for acid demineralization prior to extraction. The ability to extract proteins from bone without prior demineralization offers important advantages in efficient representative extraction of protein and significant time savings during sample preparation.