High-throughput mutagenesis approaches are widely employed to systematically characterize protein functions and play a critical role in therapeutic developments. As the largest class of membrane receptors, G protein-coupled receptors (GPCRs) are a primary focus of these studies. However, while significant progress has been made in understanding GPCRs themselves, mutagenesis studies on their ligands have lagged behind, because of the difficulties in solubilizing the target receptor. In this study, we present a novel approach that employs lipid vesicles to embed and stabilize target membrane receptors, allowing direct ligand screening. We applied this platform to investigate the anaphylatoxin complement 5a (C5a) and examined how mutations affect binding to its two native GPCRs: complement 5a receptor 1 (C5aR1) and complement 5a receptor 2 (C5aR2). The screening revealed new insights into the molecular basis of the interaction and led to the discovery of novel ligands that selectively activate C5aR2, but not C5aR1.