Reduction and alkylation are essential steps in shotgun proteomic sample preparation, but over-alkylation can occur on peptide N-terminus and amino acid residues other than cysteine, which adversely affects protein identification and quantification. To date, different reduction and alkylation conditions are used in different laboratories, but a systematic evaluation of the extent of over-alkylation from these different protocols has not been done yet. In this study, we comprehensively evaluated various sample preparation protocols that used different denaturants, different digestion buffers and different concentrations of reduction and alkylation reagents. N-term carbamidomethylation occurred on 2-11% of the identified peptides in some commonly used protocols, and it increased over 40 times as the concentration of dithiothreitol and iodoacetamide increased from 1mM/4mM to 20mM/80mM. The remaining excessive iodoacetamide was proven to be the major cause of over-alkylation during trypsin digestion. The use of digestion buffers at pH 6 reduces over-alkylation, but greatly introduces some other artificial modifications. In conclusion, it is feasible to minimize over-alkylation by using a few mM of dithiothreitol and iodoacetamide at the ratio of 1:2 for reduction and alkylation or quenching the excessive iodoacetamide with dithiothreitol before digestion. This study greatly improves proteomic sample preparation in the aspect of reduction and alkylation.