The cartilage zone of the velvet antler is richly vascularized, this being a major difference to the classical cartilage, in which there are no blood vessels. Angiogenesis and rapid growth of vasculature in velvet antler cartilage (VAC) make it an ideal model for discovering the novel angiogenic regulatory factors. However, the proteomic analysis of VAC is challenging due to the serious interference of proteoglycans (PGs) and collagens. To achieve a comprehensive proteome characterization of VAC, herein, we developed an ionic liquid-based method using 1-dodecyl-3-methylimidazolium chloride ([C12-mim]Cl) for selective extraction of cellular proteins from VAC. Compared with the previous cetylpyridinium chloride (CPC)-based method, the developed [C12-mim]Cl-based method takes much less processing time, shows facile preparation procedure and good compatibility towards downstream proteomic analysis, leading to the identification of more protein groups (1543 vs 753), membrane proteins (663 vs 279) and transmembrane proteins (217 vs 58).