The anaphylatoxin C5a is a complement activation peptide associated with many inflammatory and immune related disorders. C5a binds and activates two endogenous transmembrane receptors, C5aR1 and C5aR2. Activation of C5aR1 is known to be pro-inflammatory and is linked with many inflammatory conditions. The second endogenous receptor, C5aR2, was discovered more recently, and its functionality remains elusive. C5a is utilized for the interrogation of C5aR1 and C5aR2, and the development of next generation inhibitors for each receptor. Currently, the main sources of C5a are via isolation of endogenous C5a from human/mouse blood, or production by recombinant expression. An alternative approach to C5a production is chemical synthesis, which has several advantages including the ability to introduce non-natural amino acids and site-specific modifications, while also maintaining a lower probability of the C5a being contaminated with molecules of microbial origin or other endogenous proteins. Here we describe the efficient synthesis by SPPS of both human (hC5a) and mouse C5a (mC5a) without the need for ligation chemistry. We validate the synthetic peptides by comparing pERK1/2 signaling in CHO cells stability expressing C5aR1 (for hC5a) and RAW264.7 cells (for mC5a). Further validation is also performed by measuring β-arrestin recruitment in HEK293 cells transiently transfected with human C5aR2. We also describe the functionalization of synthetic hC5a by the introduction of a lanthanide chelating cage and demonstrate the capability to screen the binding of unlabeled ligands at C5aR1. Finally, we verify that the synthetic ligands are functionally active in vivo, and perform similar to recombinant C5a by assessing C5a-induced neutrophil migration in mice. We propose that synthetic hC5a and mC5a are valuable alternatives to recombinant C5a for in vitro and in vivo applications.