The landscape of new psychoactive substances (NPS) is constantly evolving, with new compounds entering the illicit drug market at a continuous pace. Of these, opioid NPS form a threat given their high potency and prevalence. Whereas previously, the use of fentanyl and fentanyl derivatives was the main point of attention, legislations have reacted accordingly, which may have been a driving force towards the (ab)use of alternative µ-opioid receptor (MOR) agonists. In contrast to fentanyl (analogues), details on these novel non-fentanyl opioid NPS are scarce. We investigated the biological activity of a panel of 11 'alternative', newly emerging MOR agonists (2-methyl-AP-237, AP-237, bromadol, brorphine, butorphanol, isotonitazene, mitragynine, 7-OH-mitragynine, MT-45, piperidylthiambutene and tianeptine) using two closely related in vitro MOR activation bio-assays, monitoring either G protein (mini-Gi) or β-arrestin2 (βarr2) recruitment. Activity profiles were obtained for all tested compounds, with values for potency (EC50) ranging from 1.89 nM (bromadol) to >3 µM (AP-237 and tianeptine). Bromadol, brorphine, isotonitazene, piperidylthiambutene and tianeptine had the highest efficacy (Emax) values, exceeding that of the reference compound hydromorphone ≥1.3-fold (βarr2 assay) and >2.6-fold (mini-Gi assay). Information on the recruitment of two distinct signaling molecules additionally enabled evaluation of biased agonism, none of the evaluated opioids being significantly biased. Taken together, this study is the first to systematically investigate the in vitro biological activity of a diverse panel of emerging non-fentanyl opioid NPS at MOR. Given the known danger of (fatal) intoxications with many opioid NPS, it is important to continuously monitor and characterize newly emerging compounds.