A wide range of fluorescent dyes and reagents exist for labeling organelles in live and fixed cells. Choosing between them can lead to confusion, and optimization for many of them can be challenging. Presented here is a discussion on the commercially available reagents that have shown the most promise for each organelle of interest, including endoplasmic reticulum/nuclear membrane, Golgi apparatus, mitochondria, nucleoli, and nuclei, with an emphasis on localization of these structures for microscopy. Included is a featured reagent for each structure with a recommended protocol, troubleshooting guide, and example image. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Endoplasmic reticulum and nuclear membrane labeling using ER-Tracker reagents Basic Protocol 2: Labeling Golgi apparatus using dye-labeled ceramides Basic Protocol 3: Labeling mitochondria using MitoTracker Red CMXRos Basic Protocol 4: Labeling nucleoli using SYTO RNASelect Green.