Despite the advances in modern medicine, our ability to adequately diagnose and treat a number of disease processes is still lacking. Key to our knowledge is the understanding of genetic regulation, intracellular activity, and cell-to-cell interactions. It is estimated that there are 20,000–30,000 genes that comprise the human genome code for greater than one million different proteins, any of which may be biomarkers for disease, targets for drug therapies, or insights into formulating better or more tolerable treatment strategies. With the expanding interest in basic and translational science in the field of surgery, it is important that the academic surgeon revisit the fundamentals of protein analysis to successfully formulate an experimental design. There are an endless number of ways to purify, identify, and classify the function of proteins. The major steps in protein analysis involve (1) determining the source, (2) extracting the protein, (3) purifying the protein, (4) identifying and/or quantifying the protein, and (5) establishing its function by assessing how it interacts with other proteins. This chapter reviews the current methods involved with each of these steps and acts as a guide to answering the academic surgeon’s queries.