The　purpose　of　this　study　was　to　investigate　the　site-selective　binding　of　erlotinib　hydrochloride　(ET),　a　targeted　anticancer　drug,　to　bovine　serum　albumin　(BSA)　through　1H　NMR,　spectroscopic,　thermodynamic,　and　molecular　modeling　methods.　The　fluorescence　quenching　of　BSA　by　ET　was　a　result　of　the　formation　of　BSA-ET　complex　with　high　binding　affinity.　The　site　marker　competition　study　combined　with　isothermal　titration　calorimetry　experiment　revealed　that　ET　binds　to　site　II　of　BSA　mainly　through　hydrogen　bond　and　van　der　Waals　force.　Molecular　docking　was　further　applied　to　define　the　specific　binding　site　of　ET　to　BSA.　The　conformation　of　BSA　was　changed　in　the　presence　of　ET,　revealed　by　synchronous　fluorescence,　circular　dichroism,　and　three-dimensional　fluorescence　spectroscopy　results.　Further,　NMR　analysis　of　the　complex　revealed　that　the　binding　capacity　contributed　by　the　aromatic　protons　in　the　binding　site　of　BSA　might　be　greater　than　the　aliphatic　protons.　©　2012　Taylor　and　Francis.