["Acta Physiologica, Volume 231, Issue 2, February 2021. ", "\nAbstract\n\nAims\nIn the pathogenesis of several diseases, neo‐angiogenesis is increased (e.g. tumour growth). The peptide L‐glutamyl‐L‐tryptophan (EW/IM862) has been claimed to exhibit inhibitory effects on tumour growth in vivo. However, the potential role of natural peptides with respect to anti‐angiogenic properties is unsettled. The current study explores anti‐angiogenic effects of the dipeptides WL, EW, IW and WE.\n\n\nMethods and Results\nUsing a bottom‐up strategy, we first evaluated the effects of the peptides on VEGFR‐2 signalling and quantified their effects in different angiogenesis assays. WL consistently had the strongest effects on phosphorylation of VEGFR‐2 and downstream signalling. Therefore, this peptide was chosen in comparison with EW to further assess anti‐angiogenic properties. However, sprout formation in three‐dimensional (3D) fibrin gel bead assay was significantly inhibited by EW only. Furthermore, vessel sprouting in the mouse aortic ring assay was decreased by the presence of WL and EW compared to control. Results from a chorioallantoic membrane assay showed that under vascular endothelial growth factor (VEGF) stimulation WL and EW decreased the number of blood vessels versus control. These results were in line with those obtained in a matrigel plug assay. The VEGF‐induced increase in the haemoglobin content was nearly abolished when treatment was combined with either WL or EW application. In the murine model of oxygen‐induced retinopathy, WL exhibited a small albeit significant anti‐angiogenic effect.\n\n\nConclusion\nComprehensive screening of WL suggests an anti‐angiogenic effect, demonstrated in in vitro, ex vivo and in vivo models. Thus, WL is a dipeptide with potential anti‐angiogenic effects and is worthy for further exploration.\n\n"]