Abstract: The present study investigates the postprandial serum glucose lowering mechanism of soluble and insoluble dietary fibers isolated from Cassia fistula and Tamarindus indica seed comprised with natural Guar Gum (GG) and Tragacanth (TRAG) in vitro (glucose dialysate and α-amylase activity). The study was performed by two methods that’s were effect on diffusion of glucose in glucose-dietary fiber system and effect of dietary fiber on the activity of α-amylase. The results have shown that these isolated dietary fibers diminish the postprandial glucose level more than 50% as compared to control. Tamarindus indica soluble fiber (TSF) has more inhibitory action against glucose dialysate in comparison to Cassia fistula soluble fiber (CSF), GG and TRAG. Tamarindus indica insoluble fiber (TIF) and Cassia fistula insoluble fiber (CIF) also had shown significant glucose lowering result against the control and standards. All the tested fibers had also shown the significant hindered α-amylase activity on starch as compared to control. The results of this research suggested that isolated dietary fiber could be potentially effective in the treatment of hyperglycemia in human as per other classical sources of dietary fibers.