Free fatty acid receptor 2 (FFA2, GPR43) is a G protein-coupled 7-transmembrane receptor for short chain fatty acids (SCFAs) that is implicated in inflammatory and metabolic disorders. The SCFA propionate has close to optimal ligand efficiency for FFA2, and can hence be considered as highly potent given its size. Propionate, however, does not discriminate between FFA2 and the closely related receptor FFA3 (GPR41). To identify FFA2 selective ligands and understand the molecular basis for FFA2 selectivity, a targeted library of small carboxylic acids (SCAs) was examined using holistic, label-free dynamic mass redistribution technology for primary screening, and the receptor-proximal G protein [35S]GTPγS activation assay, inositol phosphate and cAMP accumulation assays for hit confirmation. Structure-activity relationship analysis allowed formulation of a general rule to predict selectivity for SCAs at the orthosteric binding site, where ligands with substituted sp3-hybridized α-carbons preferentially activate FFA3, while ligands with sp2- or sp-hybridized α-carbons prefer FFA2. The orthosteric binding mode was verified by site-directed mutagenesis: replacement of orthosteric site arginine residues by alanine in FFA2 prevented ligand binding, whilst molecular modeling predicted the detailed mode of binding. Based on this, selective mutation of three residues to their non-conserved counterparts in FFA3 was sufficient to transfer FFA3 selectivity to FFA2. Thus, selective activation of FFA2 via the orthosteric site is achievable with rather small ligands, a finding with significant implications for the rational design of therapeutic compounds selectively targeting the SCFA receptors.