Proteins are complex molecular entities with different sizes, molecular structures, and physical and chemical properties. Protein characterization is one of the important parts of protein expression. Characterizing a protein can obtain useful information on its identity, purity, structure, conformation, and activity. The precise molecular weight, amino acid sequence, charge variances, aggregation level, glycosylation, and oxidation level are the key to the in-depth characterization of proteins. Different post-translational modifications, highly specific three-dimensional structures, and the potential for aggregation, adsorption, and truncation determine the complexity of proteins. No one analytical platform or technique can meet all the requirements to characterize proteins so far.