Capillary isoelectric focusing (cIEF) is a very powerful separation technique. However, successful implementation of this technique in various fields of science requires a few prerequisites including a repeatable formation of pH gradient and a set of internal standards (i.e., markers of isoelectric point) to track the actual profile of the pH gradient. Overview of the available isoelectric point markers and their syntheses is presented in this review. Moreover, recent applications of cIEF for analysis of complex larger structures such as viruses, vesicles, and bacterial cells are also reviewed.