G protein-coupled receptors (GPCRs) are the largest family of integral membrane proteins and are targets for more than 25% of prescription drugs. Importantly, there are numerous sub-families of the GPCRs that are activated by the same endogenous agonists but modulate different physiological pathways. To obtain subtype specific ligands is therefore essential to target specific GPCRs and minimize side effects. Such an example are the α1A- and α1B-adrenoceptors (α1A-AR and α1B-AR), which may drive opposing responses to adrenaline or noradrenaline activation in modulating the cardiovascular and nervous systems and therefore present an interesting challenge to find novel subtype selective ligands. While there has been substantial technical progress in solving the crystal structures of GPCRs, applying solution-based methodologies, such as NMR and SPR, has remained problematic. These methods are ideal for searching fragment and compound libraries for novel weak binding drug ligands, but require the GPCR to be stable in the apo-state and in a detergent system for long periods of time. Using a novel stabilization technique (CHESS) α1A-AR and α1B-AR have been engineered to meet these criteria. Here we used detergent-stabilized α1A-AR and α1B-AR variants to probe the specific binding of the endogenous agonist adrenaline and the peptide allosteric modulator ρ-TIa using Saturation Transfer Difference NMR (STD NMR), a powerful method for characterizing ligand-protein interactions, drug screening and epitope mapping. The results enabled us to determine the molecular basis for the subtle receptor sub-type selectivity differences of the two ligands. As GPCRs are the largest, yet underexploited class of drug targets, this work has broad applications for studying GPCR-ligand interactions and for drug design and discovery.