The 77-residue peptide C3a and its receptor C3aR are part of the complement system. C3a is a potent mediator of inflammation and has been implicated in numerous disease pathways, including asthma, rheumatoid arthritis and allergies, and is therefore a prime target for therapeutic intervention. Because C3a is rapidly degraded in serum, efforts have focused on designing small, stable C3aR agonists. However, the development of synthetic ligands has been hampered by the lack of information regarding binding characteristics of the C3a:C3aR complex. Here we investigate the structure/function relationship of a series of peptide agonists based on the sequence of the C3aR agonist EP67, leading to the development of two optimised peptides showing increased potency. Mixture-based positional scanning libraries were generated to probe the effect of a large range of amino acid substitutions at several positions. This allowed profiling of side chain characteristics associated with an increase in activity at each position examined. Activity data from library screens facilitated the design and synthesis of our two optimised peptides, containing residue substitutions identified as being highly active in the library screens. These two peptide analogues showed increased potency compared to EP67 in calcium flux assays with U937 cells.