G-protein-coupled receptors (GPCRs) constitute the largest family of transmembrane proteins. They control almost all physiological processes in humans and consequently emerge as a molecular target of around 40% of the commercially available drugs. However, most of the GPCRs subtypes are still unexplored, mostly due to the lack of specific ligands. Since the number of bioactive compounds found in animal venoms can reach up to 40 millions, the use of this natural library as a source for new ligands and pharmacological tool discoveries comes to light. This work aimed the development and the improvement of a new MS based technique able to identify new ligands for GPCRs. Vasopressin type 2, human Endothelin (ET-B) and human Muscarinics M1 receptors were incubated with their respective ligands (vasopressin, endothelin-1, and MT-7 toxin) plus a simple mix of known peptides. After the incubation, two fractions were obtained: one containing the peptides that bound to the receptors (‘bound fraction’) and the second, the peptides that did not bind (‘free fraction’). Both fractions were analyzed with a MALDI-TOF/TOF. The second phase of this work is linked to the incubation of receptors with crude (or fractions of) animal venom to ensure the workability of the protocol when applied for a complex mixture of unknown compounds and to discover new specific ligands. The MS analyses showed the presence of the non-specific peptides in the free fraction, while the ligands were just detected in the bound fraction. These results demonstrate the feasibility of the protocol in fishing for specific ligands. The presented work developed and improved a new methodology to screen for specific ligands for GPCR in either a simple mixture or a complex mixture; in consequence, this may assist the discovery of new ligands or even characterize orphan receptors, since the protocol can be adapted for other GPCR.