Abstract: Deer antler, a valued traditional Chinese medicine, faces persistent challenges in quality control due to the complex species origins, prevalent adulteration practices, and the absence of specific quantitative standards. To address this challenge, a proteomics strategy was employed to screen signature peptides from deer antlers and reindeer antlers in our pevious study. Combining the reported antler peptides from Cervidae, a comprehensive specificity revalidation was conducted. Finally, six deer antler signature peptides were identified as quality control (QC) markers for quantitative analysis, while reindeer, cow, sheep, and pig-specific peptides were determined as markers for adulteration inspection. Based on these QC markers, an ultrasonic-assisted enzymatic digestion method that completed sample preparation within 6 hours was developed in this study, significantly enhancing analytical efficiency. A UHPLC-MS/MS was utilized for establishment of absolute quantification and adulteration inspection method for deer antlers. The results indictated the validated methods showed high sensitivity and specificity, meeting all requirements for methodological requirements. The proposed method was subsequently applied to 56 commercially available samples (including 28 batches of deer antler slices and 28 batches of deer antler powder). The results revealed that 17.9% for slices and 10.7% for powder were deemed authentic and unadulterated. The total detection rate for the ingredients of reindeer was 73.2% and some samples were found to contain cattle-derived ingredient, indicating the rampant chaos in the deer antler market. This study provided a comprehensive and reliable technical approach to address the aforementioned challenges and achieve quality control for deer antler and its related preparations. The summarized strategy for screening species-spcific peptides and developing quality control methods would provide significant references on the study of quality standards for animal-derived traditional Chinese medicines.