Immunogenicity is a major concern in drug development as anti-drug antibodies in many cases affect both patient safety and drug efficacy. Another concern is often the limited half-life of drugs, which can be altered by different chemical modifications, like acylation with fatty acids. However, acylation with fatty acids has been shown in some cases to modulate T cell activation. Therefore, to understand the role of acylation with fatty acids on immunogenicity we tested three immunogenic non-acylated peptides and 14 of their acylated analogues for binding to 26 common HLA class II alleles, and their ability to activate T cells in an ex vivo T cell assay. Changes in binding affinity associated with acylation with fatty acids were typically modest, though a significant decrease was observed for influenza HA acylated with a stearic acid, and affinities for DQ alleles were consistently increased. Importantly, we showed that for all three immunogenic peptides acylation with fatty acids decreased their capacity to activate T cells, a trend particularly evident with longer fatty acids typically positioned within the peptide HLA class II binding core region, or when closer to the C-terminus. With these results we have demonstrated that acylation with fatty acids of immunogenic peptides can lower their stimulatory capacity, which could be important knowledge for drug design and immunogenicity mitigation.