Following the successful development of the first once-daily GLP-1 analog liraglutide we aimed to discover analogs suitable for once-weekly dosing through acylation with fatty acids, enabling binding to serum albumin in vivo. This technology allows a drug molecule that is structurally near human GLP-1, which is not possible with fusion protein strategies. To go from once-daily to once-weekly dosing, the aim of our studies were to increase albumin affinity and obtain full stability against DPP-4 degradation, without compromising a high GLP-1 receptor (GLP-1R) affinity. The human GLP-1R was used for testing of receptor and albumin affinity. Pharmacokinetic properties were investigated in rats and pigs. Alterations in the peptide sequence as well as the fatty acid “sidechain” were explored. The latter was the key feature to secure the combination of high albumin affinity and high GLP-1R potency. Length and type of fatty acid as well as the linking chemistry between the fatty acid and GLP-1 were the parameters investigated. Semaglutide was selected as the optimal once weekly candidate. Semaglutide has two amino acid substitutions compared to human GLP-1 (Aib(8), Arg(34)) and is derivatized at lysine 26. The GLP-1R affinity of semaglutide (0.38 ± 0.06 nM) was three-fold decreased compared to liraglutide, whereas the albumin affinity was increased. The plasma half-life was 46.1 h in mini-pigs following i.v. administration, and semaglutide has an MRT of 63.6 h after s.c. dosing to mini-pigs. Semaglutide is currently in phase 3 clinical testing.