Ciprofloxacin (CPFX) and Tetracycline (TC) are two of the most widely used antibiotics in human and veterinary medicines. Their existence in the environment has experienced much attention because of the potential adverse effects on humans and ecosystem functions. In this paper, the interaction mechanism between the two antibiotics and Human holo transferrin by spectroscopic and molecular docking methods. The distance r, between the antibiotics and the protein was obtained according to FRET which pointed at a successful formation of a drug – protein complex. Molecular docking study provided possible binding sites of antibiotics within the proteins. The obtained data can be convenient for determining usage drug doses in drug delivery. Keywords: Human holo transferrin, ciprofloxacin, Tetracycline, FRET, Molecular Docking.