Targeting vascular endothelial growth factor receptor (VEFGR) and its co–receptor neuropilin–1 (NRP–1) is an interesting vascular strategy. tLyp–1 is a tumor–homing and penetrating peptide of 7 amino acids (CGNKRTR). It is a truncated form of Lyp–1 (CGNKRTRGC), which is known to target NRP–1 receptor, with high affinity and specificity. It is mediated by endocytosis via C–end rule (CendR) internalization pathway. The aim of this study is to evaluate the importance of each amino acid in the tLyp–1 sequence through alanine–scanning (Ala-scan) technique, during which each of the amino acid in the sequence was systematically replaced by alanine to produce 7 different analogues. In silico approach through molecular docking and molecular dynamics are employed to understand the interaction between the peptide and its analogues with the NRP-1 receptor, followed by in vitro ligand binding assay study. The C-terminal Arg is crucial in the interaction of tLyp-1 with NRP-1 receptor. Substituting this residue dramatically reduces the affinity of this peptide which is clearly seen in this study. Lys-4 is also important in the interaction, which is confirmed via the in vitro study and the MM-PBSA analysis. The finding in this study supports the CendR, in which the presence of R/K-XX-R/K motif is essential in the binding of a ligand with NRP-1 receptor. This presented work will serve as a guide in the future work pertaining the development of active targeting agent towards NRP-1 receptor.