β‐Arrestins are implicated in desensitization of activated G protein‐coupled receptors (GPCRs) and transduction of signals in their own right. Biased ligands possessing different efficacies in activating different G protein‐dependent and β‐arrestin‐dependent signals downstream of a single GPCR have been described. Here, we report the characterization of agonist bias for compounds developed along a drug discovery project of β2‐adrenoceptor agonists. Compounds, including derivatives of fenoterol, 2‐amino‐1‐phenylethanol and 2‐amino‐2‐phenylethanol, were obtained or synthesized and their effects on cardiomyocyte contractility, cAMP synthesis and β‐arrestin recruitment were determined in β2‐adrenoceptor‐expressing cells. Gs versus β‐arrestin bias were analyzed by the operational model. Gs versus Gs/Gi bias were studied by means of sensitivity towards pertussis toxin, a Gi disruptor. Three compounds with a core structure of 5‐(1‐amino‐2‐hydroxyethyl)‐8‐hydroxyquinolin‐2(1H)‐one were identified as β‐arrestin‐biased β2‐adrenoceptor agonists. These findings would facilitate the development of novel drugs for the treatment of heart failure and asthma.Support or Funding InformationThis work was supported by the Ministry of Science and Technology of the People's Republic of China under the National Key Research and Development Program of China [2018YFA0507603], the National Science and Technology Major Project [2013ZX09507001‐001002, 2013ZX09301305‐001, 2013ZX09508104, 2018ZX09739009], the National Basic Research Program [2012CB518000], the National Natural Science Foundation of China [81673355, 81872752, 31521062, 81630008, 81790621], Beijing Municipal Science & Technology Commission [Z171100000417006], and the intramural research program of the NIH, USA.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.