The development of multifunctional gold nanoparticles (AuNPs) underwent an explosion in the last two decades. However, many questions regarding detailed surface chemistry and how they are affecting the behaviors of AuNPs in vivo and in vitro still need to be addressed before AuNPs can be widely adapted into clinical settings. In this work, radioactivity analysis was employed for quantitative evaluation of I-125 radiolabeled AuNPs uptakes by cancer cells. Facilitated with this new method, we have conducted initial bioevaluation of surfactant-free AuNPs produced by femtosecond laser ablation. Cellular uptake of AuNPs as a function of the RGD density on the AuNP surface, as well as a function of time, has been quantified. The radioactivity analysis may shed light on the dynamic interactions of AuNPs with cancer cells, and help achieve optimized designs of AuNPs for future clinical applications.