Background: The processing of Chinese materia medica is an important part for the preparation of traditional Chinese medicine (TCM) which has been believed that can change the chemical composition and physiological function. Velvet antler is a kind of precious TCM and widely used after hot processing as folk medicines in China. However, no strategy has been presented to reveal the processing principle of velvet antler. Objective: The aim of this study is to develop a method for exploring the mechanism of hot processing on velvet antler. Materials and Methods: In this study, the chemical compositions of fresh velvet antler (FVA) and hot processed velvet antler (HPVA) were compared based on the 1H nuclear magnetic resonance (1H NMR) and multivariate statistical analysis methods to explore the mechanism of hot processing on velvet antler. Results: It showed that hot processing increased phosphorylcholine, taurine, alanine, uridine, phenylalanine, uracil, tyrosine, accompanied by decreased bile acid, choline, lipid 2 and succinic acid in comparison to the FVA, which changed significantly in 52 common metabolites of FVA and HPVA. In addition, a clear separation between FVA and HPVA was obtained by using principal component analysis and 11 “chemical markers” were found by using orthogonal partial least squares discriminant analysis (OPLS-DA) approaches. Particular attention was given to the “chemical markers” with potentially beneficial bioactivities. Conclusion: The study demonstrated that 1H NMR-based chemometric approach could be a promising tool for investigation of the hot processing of velvet antler in a holistic way of TCM.