Fluorescence microscopy techniques have been widely adopted in biology for their ability to visualize the structure and dynamics of a wide range of cellular and subcellular processes. The specificity and sensitivity that these techniques afford have made them primary tools in the characterization of protein localizations within cells. Many of the fluorescence microscopy techniques require cells to be fixed via chemical or alternative methods before being imaged. However, some fixation methods have been found to induce the redistribution of particular proteins in the cell, resulting in artifacts in the characterization of protein localizations and functions under physiological conditions. Here, we review the ability of commonly used cell fixation methods to faithfully preserve the localizations of proteins that bind to chromatin, undergo liquid-liquid phase separation (LLPS), and are involved in the formation of various membrane-bound organelles. We also review the mechanisms underlying various fixation artifacts and discuss potential alternative fixation methods to minimize the artifacts while investigating different proteins and cellular structures. Overall, fixed-cell fluorescence microscopy is a very powerful tool in biomedical research; however, each experiment demands the careful selection of an appropriate fixation method to avoid potential artifacts and may benefit from live-cell imaging validation.