The glucagon-like peptide 1 receptor (GLP-1R) is a class B G-protein coupled receptor (GPCR) and diabetes drug target expressed mainly in pancreatic β-cells that, when activated by its agonist glucagon-like peptide 1 (GLP-1) after a meal, stimulates insulin secretion and β-cell survival and proliferation. The N-terminal region of GLP-1 interacts with membrane-proximal residues of GLP-1R, stabilizing its active conformation to trigger intracellular signaling. The best-studied agonist peptides, GLP-1 and exendin-4, share sequence homology at their N-terminal region; however, modifications that can be tolerated here are not fully understood. In this work, a functional screen of GLP-1 variants with randomized N-terminal domains reveals new GLP-1R agonists and uncovers a pattern whereby a negative charge is preferred at the third position in various sequence contexts. We further tested this sequence-structure-activity principle by synthesizing peptide analogues where this position was mutated to both canonical and noncanonical amino acids. We discovered a highly active GLP-1 analogue in which the native glutamate residue three positions from the N-terminus was replaced with the sulfo-containing amino acid cysteic acid (GLP-1-CYA). The receptor binding and downstream signaling properties elicited by GLP-1-CYA were similar to the wild type GLP-1 peptide. Computational modeling identified a likely mode of interaction of the negatively charged side chain in GLP-1-CYA with an arginine on GLP-1R. This work highlights a strategy of combinatorial peptide screening coupled with chemical exploration that could be used to generate novel agonists for other receptors with peptide ligands.