Protein extraction methods [urea, trichloroacetic acid (TCA), and acetic acid] were compared for protein recovery from rat incisor developing enamel in the S phase (intermediate/late secretion), M1 phase (early maturation), M2 phase (intermediate maturation), and M3 phase (final maturation). We compared the protein recoveries with the percentage of enamel matrix dry weight burnt off by incineration. Our results indicate that TCA and urea were equally efficient for the extraction of S-stage proteins (85% and 90% recovery, respectively), while urea was the best for M1-stage proteins (92% recovery), and TCA the best for M2-stage (99% recovery) and M3-stage (60% recovery) proteins. The other methods yielded less than 30% recovery in comparison to incineration for M2 and M3 stages. The fact that urea extraction works well in the S and M1 stages and not thereafter is probably related to the changes in the proteins during enamel development and the amount of mineral that needs to be dissolved. TCA is the single method that effectively recovered proteins from all developmental stages of the rat incisor enamel.