Although immobilization of growth factors (GFs) on culture substrates has been investigated as a cell culture technique, quantification of the bioactive stability of immobilized GFs has not been studied in detail. We developed a system of measuring GF stability using heparin-immobilized collagen substrate, vascular endothelial growth factor (VEGF). and human umbilical vein endothelial cells (HUVECs). VEGF solution was added to a heparin-crosslinked substrate and immobilized on the substrate. HUVECs were cultured on the VEGF-immobilized substrate, and the mitochondrial activity of the cells was assessed. A calibration curve showing the relation between HUVEC mitochondrial activity and immobilized VEGF density was constructed. Next, immobilized VEGF and VEGF solution were pre-incubated at 37°C, and the pre-incubated VEGF was added to heparin-crosslinked substrate. HUVEC mitochondrial activity declined as the pre-incubation period increased. Density of active immobilized VEGF was derived from HUVEC mitochondrial activity using the calibration curve, and the effect of pre-incubation of VEGF at 37°C was demonstrated quantitatively. Even after culture for 16 days, immobilized VEGF in culture medium at 37°C retained 43%of the initial bioactivity. Immobilized VEGF retained activity better than VEGF solution for at least 12 days of pre-incubation. The present results indicate that immobilization improves the stability of VEGF.