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Abstract Developing improved fluorescent probes for imaging the endoplasmic reticulum (ER) is necessary for structure‐activity studies of this dynamic organelle. Two coumarin‐based compounds with sulfonamide side groups were synthesized and characterized asER‐targeting probes. Their selectivity to target theERin HeLa andGM07373 mammalian cells was shown with co‐localization experiments using commercially available probes that localize in theER, mitochondria, or lysozymes. The hydrophobicity of the coumarin‐based probes was comparable to known probes that partition into theERmembrane. Their cytotoxicity in mammalian cells was low withIC50 values that range from 205 to 252 μm. The fluorescent quantum yields of the coumarin‐based probes when excited with 400 nm light were 0.60, and they have a much narrower emission spectrum (from 435 to 525 nm in methanol) than that of the only commercially availableERprobe that is exited with 400 nm light (ER‐Tracker™ Blue‐WhiteDPX). Thus, the coumarin‐based probes are more useful for multicolor imaging with yellow and red emitting fluorophores. In addition to the above benefits,ERlabeling was achieved with the coumarin‐based probes in both live cells and fixed cells, revealing their versatility for a wide range of cellular imaging applications.