Background: Three-dimensional (3D) biomimetic models via various approaches can be used by therapeutic applications of tissue engineering. Creating an optimal vascular microenvironment in 3D model that mimics the extracellular matrix (ECM) and providing an adequate blood supply for the survival of cell transplants are major challenge that need to be overcome in tissue regeneration. However, currently available scaffolds-depended approaches fail to mimic essential functions of natural ECM. Scaffold-free microtissues (SFMs) can successfully overcome some of the major challenges caused by scaffold biomaterials such as low cell viability and high cost. Methods: Herein, we investigated the effect of soluble integrin binding peptide of arginine-glycine-aspartic acid (RGD) on vascularization of SFM spheroids of human umbilical vein endothelial cells.In vitro-fabricated microtissue spheroids were constructed and cultivated in 0 mM, 1 mM, 2 mM, and 4 mM of RGD peptide. The dimensions and viability of SFMs were measured. Results: Maximum dimension and cell viability observed in 2 mM RGD containing SFM. Vascular gene expression of 2 mM RGD containing SFM were higher than other groups, while 4 mM RGD containing SFM expressed minimum vascularization related genes. Immunofluorescent staining results indicating that platelet/endothelial cell adhesion molecule and vascular endothelial growth factor protein expression of 2 mM RGD containing SFM was higher compared to other groups. Conclusion: Collectively, these findings demonstrate that SFM spheroids can be successfully vascularized in determined concentration of RGD peptide containing media. Also, soluble RGD incorporated SFMs can be used as an optimal environment for successful prevascularization strategies.