Proteins are essential biomolecules for use in research, diagnostics, and therapy; however, toxicity and side effects, lack of activity and specificity of the manufactured proteins arise due to altered properties and factors derived from the complexity of their structures, cellular specificity of action, dynamic folding, and reactivity. In vivo, proteins perform diverse functions and may play multiple roles, such as enzymatic activity coupled with genetic regulation, membranes traffic and rearrangements, molecular transport or transmission, among many others. Reactive amino acid residues on the protein surfaces participate in signal transduction pathways via cascades of reversible interactions and redox reactions. In isolated form, proteins lose the support of the native environment, resulting in loss of functional states. The replacement of the cellular compounds by appropriate organic and inorganic molecules is critical during protein production to keep them in a homogeneous state that is as close as possible to that of the native system. In addition, the formation of homo-oligomers may inactivate proteins, triggering immune responses in the targeted cells. It is challenging to identify the multiple factors that can affect protein properties during the entire process of production from cloning to manufacturing. The fast growth of bioinformatic tools and databases allow statistical evaluation of a large variety of those critical factors and how they contribute to the manufacturing process. By applying innovative multidisciplinary and design of experiments approaches, a rapid reduction of the variables is achievable and thus can bring about results that are more consistent to improve the quality of the manufactured proteins and the success in clinical trials. The review article provides examples of enzyme and antibody production for use in research, diagnostics, and drug discovery.