AbstractFluorescent ligand technologies have proved to be powerful tools to improve our understanding of ligand‐receptor interactions. Here we have characterized a small focused library of nine fluorescent ligands based on the highly selective β2‐adrenoceptor (β2AR) antagonist ICI 118,551. The majority of fluorescent ICI 118,551 analogs had good affinity for the β2AR (pKD >7.0) with good selectivity over the β1AR (pKD <6.0). The most potent and selective ligands being 8c (ICI 118,551‐Gly‐Ala‐BODIPY‐FL‐X; β2AR pKD 7.48), 9c (ICI 118,551‐βAla‐βAla‐BODIPY‐FL‐X; β2AR pKD 7.48), 12a (ICI 118,551‐PEG‐BODIPY‐X‐630/650; β2AR pKD 7.56), and 12b (ICI 118,551‐PEG‐BODIPY‐FL; β2AR pKD 7.42). 9a (ICI 118,551‐βAla‐βAla‐BODIPY‐X‐630/650) had the highest affinity at recombinant β2ARs (pKD 7.57), but also exhibited significant binding affinity to the β1AR (pKD 6.69). Nevertheless, among the red fluorescent ligands, 9a had the best imaging characteristics in recombinant HEK293 T cells and labeling was mostly confined to the cell surface. In contrast, 12a showed the highest propensity to label intracellular β2ARs in HEK293 T cell expressing exogenous β2ARs. This suggests that a combination of the polyethylene glycol (PEG) linker and the BODIPY‐X‐630/650 makes this ICI 118,551 derivative particularly susceptible to crossing the cell membrane to access the intracellular β2ARs. We have also used these ligands in combination with CRISPR/Cas9 genome‐edited HEK293 T cells to undertake for the first time real‐time ligand binding to native HEK293 T β2ARs at low native receptor expression levels. These studies provided quantitative data on ligand‐binding characteristics but also allowed real‐time visualization of the ligand‐binding interactions in genome‐edited cells using NanoBRET luminescence imaging.