Electrospray ionization (ESI) mass spectrometry is a powerful and versatile tool for proteomic analysis. By understanding how proteins and peptides behave during ESI, it is possible to predict source conditions that will maximize ionization efficiency, ultimately leading to lower detection limits for protein identification and more accurate quantitation. In this article, we provide an overview of a variety of electrospray-based ionization methods, including nanospray, liquid chromatography and capillary electrophoresis-coupled sources, and how they are optimized for proteomic samples. We will touch upon analyte characteristics, solvent/eluent conditions as well as optimization of ESI for top-down, bottom-up and quantitative experiments.