The aim of the present research work was to design Tween-80 coated silk fibroin nanoparticles of doxorubicin (DOX) and it's radiolabeling with Technetium-99 m (^99mTc). Radiolabeling could be used as a tool to analyze the efficacy of design system through cellular uptake studies. The DOX loaded silk fibroin nanoparticles (SFN) of ~100 nm size were prepared by using desolvation method. Radiolabeling of DOX, and nanoparticles was performed with ^99mTc by using stannous chloride. The maximum labeling efficiency of more than 90% was found to be at 1:10 ratio of stannous chloride and DOX at pH 5.5 for 30 min incubation time. The product found to be stable in serum, saline and for transchelation study (less than 5%). Cellular uptake studies showed the higher uptake of radiolabeled formulation of Tween-80 coated silk fibroin nanoparticles (TSFN) i.e 1.16 times higher than plain SFN and 1.62 times higher than free drug for C-6 cell line and 1.18 times higher than plain SFN and 2.10 times higher than free drug for LN-229. The higher uptake of TSFN by cancer cell lines may provide the opportunity to these modified SFN to be used as a vector for the delivery of drugs to the brain.