A　method　was　established　to　determine　the　activity　of　hexokinase　by　reverse-phase　high-performance　liquid　chromatography　(RP-HPLC).　The　activity　of　hexokinase　was　determined　by　detecting　the　amount　of　ADP　in　the　reaction　that　converts　glucose　into　glucose-6-phosphate.　The　separation　degree　of　ATP　and　ADP　was　as　good　as　3.46　(separation　degree　＞1.5).　In　the　range　of　0.01-0.　40　mmol/L,　there　was　a　good　linear　relationship　between　concentration　of　ADP　and　its　peak　areas　(the　correlation　coefficient　was　0.999).　The　relative　standard　deviation(RSD)　of　intraday　was　1.43%-1.　46%,　that　of　interday　was　2.12%-2.15%.　At　30　°C　(optimal　temperature)　and　pH7.0　(optimal　pH),　the　recovery　rate　of　hexokinase　was　95.3%-97.6%.　The　results　showed　that　the　method　had　good　precision　and　high　recovery　rate,　and　the　enzyme　activity　was　determined　through　only　one-step　reaction.　To　some　extent,　this　method　can　reduce　the　cost.　©　2011　Wuhan　University　and　Springer-Verlag　Berlin　Heidelberg.