Purpose: This study aimed to compare between the effect of chitosan and GC MI paste plus on re-mineralization of demineralized enamel in-vitro. Subjects and methods: Extracted upper premolars from orthodontic patients. Sample premolars were examined one by scanning electron microscope with energy dispersive X-ray spectroscopy to determine the Ca/P ratio before demineralization and the other by Fourier transform Infrared spectroscopy to determine the degree of conversion of bonds. After demineralization, two premolars samples were selected; one was examined by scanning electron microscope with energy dispersive X-ray spectroscope and the other was examined by FTIR to determine the degree of conversion of bonds. The remaining premolars were divided equally into 3 groups: control group (GI) premolars were remineralized using artificial saliva only for 14 days, (GII) premolars were re-mineralized by chitosan hydrogel for 14 days and (GIII) premolars were re-mineralized by GC MI paste plus for 14 days. Results: They were 46 premolars and after both negative and positive control, they were divided into 3 groups 14 premolar each where, GI showed least re-mineralization occurred, where the peripheries of prisms were lost. GII chitosan group showed the most re-mineralized results as it was not only increase in minerals but also thickness of enamel by addition of minerals' layer on the surface and GIII GC MI paste plus showed average re-mineralization process. Statistical results: GII showed the highest mean % of re-mineralization followed by GIII and then GI. Statistical difference was significant. Conclusion: Chitosan is better re-mineralizing agent than GC MI paste plus.