Somapacitan is a long-acting, once-weekly, albumin-binding growth hormone (GH) derivative. The reversible albumin-binding properties leads to prolonged circulation half-life. Here, we investigated and compared somapacitan with human GH on downstream receptor signaling in primary hepatocytes and hepatocellular models and using isothermal titration calorimetry to characterize receptor binding of somapacitan in the presence or absence of human serum albumin (HSA). With non-invasive fluorescence imaging we quantitatively visualize and compare the temporal distribution and examine the tissue-specific growth hormone receptor (GHR) activation at distribution sites. We found that signaling kinetics were slightly more rapid and intense for GH compared with somapacitan. Receptor binding isotherms were characterized by a high and a low affinity interaction site with or without HSA. Using in vivo optical imaging we found prolonged systemically biodistribution of somapacitan compared with GH, which correlated with plasma pharmacokinetics. Ex vivo mouse organ analysis revealed that the temporal fluorescent intensity in livers dosed with somapacitan was significantly increased compared with GH-dosed livers and correlated with the degree of downstream GHR activation. Finally, we show that fluorescent-labeled analogs distributed to the hypertrophic zone in the epiphysis of proximal tibia of hypophysectomized rats and that somapacitan and GH activate the GHR signaling in epiphyseal tissues.