Abstract: Objective：To analyze different protein extraction methods from tooth germ, and find a suitable protein extraction method for high through-put proteome analysis of tooth development. Methods：The deciduous third molars were collected from miniature pig on embryonic 50 day and embryonic 60 day. The matrix samples of partial calcification tooth germ were fractionated with 50 mM carbonate buffer (pH 10.8). The protein extraction effect was observed by sodium dodecylsulphate–polyacrylamide gelelectrophoresis (SDS-PAGE), pulverized methods under liquid nitrogen into fine powder or with an electro-grinding machine were compared. Then proteins were quantified with the method of bicinchoninic acid (BCA), RIPA lysis buffer and 8 mol/L urea were compared. Results：SDS-PAGE showed that electro-grinding got higher quantity of tooth germ proteins than pulverizing under liquid nitrogen. At developing stages of tooth germ in miniature pigs, the extraction quantity of tooth germ proteins raised obviously when 8 mol/L urea replaced RIPA lysis buffer. Conclusions：The electro-grinding method was better for tissue utilization and 8 mol/L urea solution was suitable for extracting proteins from tooth germ with higher value of protein concentrations. The successful establishment of the extraction technique lays the foundation for the analysis methods in down- stream proteomics study.