The use of peptide ligand modified PEGylated liposomes has been widely investigated for tumor targeting. Peptides are mainly inserted in the liposomal lipid bilayer using PEG2K-lipid spacer (Peptide-PEG2K-DSPE). However, a lower cellular uptake from longer nonlinear PEG2K spacer was reported, we here synthesized a high functionality and quality (HFQ) lipid with a short, linear serine-glycine repeated peptide [(SG)5] spacer. The objective of the current study is to develop novel octaarginine (R8) peptide-HFQ lipid grafted PEGylated liposomes for glioma cells targeting. In vitro liposomes characterization showed that the mean particle size of all liposomal formulations was in the nano-scale range < 120 nm, with a small PDI value (i.e. ∼0.2) and had a spherical shape under Transmission Electron Microscope, indicating a homogenous particle size distribution. The flow cytometry in vitro cellular association data with U251 MG and U87 cells revealed that 1.5% R8-(SG)5-lipid-PEGylated liposomes exhibited significantly higher cellular association of ∼15.87 and 7.59-fold than the conventional R8-PEG2K-lipid-PEGylated liposomes (10.4 and 6.19-fold), respectively, relative to the unmodified PEGylated liposomes. Moreover, intracellular distribution studies using confocal laser scanning microscopy (CLSM) corroborated the results of the in vitro cell association. The use of ligand-HFQ-lipid liposomes could be a potential alternative to ligand-PEG2K-lipid-modified liposomes as a drug delivery system for tumor targeting. Copyright © 2021 American Pharmacists Association. Published by Elsevier Inc. All rights reserved.