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Purpose : The objective of this study is to investigate the effect of Venom Nerve Growth Factor (vNGF), isolated from the venom of Naja naja atra, on Axon Length of in vitro serum-free cultured retinal ganglion cells in rats.Methods : Neonatal rat retinal ganglion cells were cultured in vitro with various concentrations of vNGF (50ng/ml, 100ng/ml, 200ng/ml, and 300ng/ml) under serum-free conditions. Measurements of RGCs axons were obtained at two periods; the measurements of the first period were taken on the 3rd day of culture, while those of the second period were taken on the 6th day of culture. ImageJ Software was used for measurement of axon length of RGCs. One-Way ANOVA and LSD test were applied to statistically analyze the data. Scanning electron microscopy (SEM) and immunocytochemistry with antibodies to growth associated protein-43 (GAP-43) and TAU protein were used to identify retinal ganglion cells.Results : Measurements of axonal length at different concentrations during the first period did not reveal statistical significance (P=0.9>0.05) while those of the second period did disclose a statistical significance (P=0.01<0.05) . Immunocytochemical staining to the antibodies of GAP-43 and Tau protein expressed positively and SEM provided a clear structural image of RGCs.Conclusion : This study demonstrated the possibility of vNGF in promoting the axonal length of the RGCs at specific concentartion. It determined the best concentration of vNGF against in vitro serum-free cultured rat retinal ganglion cells and also explained the prohibition of the growth of RGCs axon which might result from the toxicity induced by high concentrations of vNGF.