Molecular weight (MW) analysis is crucial in protein characterization, especially when analyzing recombinant proteins or ensuring the quality and integrity of biopharmaceuticals. The analyzed intact protein mass may match perfectly with the theoretical MW; otherwise, modifications could have occurred on the primary protein structure, or the sample is a mixture. We can determine the protein’s N- and C-terminal sequences, and even the related modifications, by combining MW analysis and the Top-Down approach. Not only can MW analysis confirm the protein identity within a sample, but it can also determine the existence of impurities and obtain information about post-translational modifications (PTMs). Additional molecular features could be determined by preparing the samples differently (with or without deglycosylation), such as the mass of protein subunits and the extent of glycosylation. MW analysis is critical in confirming biopharmaceutical qualities and is often the first step to analyze glycosylated proteins, such as monoclonal antibodies.