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Proteomics is the field of study that includes the analysis of proteins, from either a basic science prospective or a clinical one. Proteins can be investigated for their abundance, variety of proteoforms due to post-translational modifications (PTMs), and their stable or transient protein–protein interactions. This can be especially beneficial in the clinical setting when studying proteins involved in different diseases and conditions. Here, we aim to describe a bottom-up proteomics workflow from sample preparation to data analysis, including all of its benefits and pitfalls. We also describe potential improvements in this type of proteomics workflow for the future.