The increasing number of G protein-coupled receptor (GPCR) crystal structures offers new opportunities for histamine receptor homology modeling. However, computational prediction of ligand binding modes in GPCRs such as the histamine H₄ receptor (H₄R), a receptor that plays an important role in inflammation, remains a challenging task. In the current work we have combined complementary in silico receptor modeling approaches with in vitro ligand structure-activity relationship (SAR) and protein site-directed mutagenesis studies to elucidate the binding modes of different ligand classes in H₄R. By systematically considering different H₄R modelling templates, ligand binding poses, and ligand protonation states in combination with docking and MD simulations we are able to explain ligand-specific mutation effects and subtle differences in ligand SAR. Our studies confirm that a combined theoretical and experimental approach represents a powerful strategy to map ligand-protein interactions.