Abstract: Method for separation and determination of o-cresol,m-cresol and p-cresol by HPLC with pre-column derivatization with acetic anhydride as derivatizing agent was proposed.Sample containing isomers of cresol was mixed with acetic anhydride with mass ratio of 1 to 8,and refluxed under heating for 2 h,giving values of yield of 98.1%,99.0% and 98.5% for o-cresyl acetate,m-cresyl acetate and p-cresyl acetate respectively.The Eclipse XDB-C18 column was used as the separation column,and a mixture of methanol and water (35+65,by vol.) was used as eluent in the separation.UV-detection at 260 nm was used in the determination.Linear relationships were kept between values of peak area and concentration in the range of (0.05-3.75)×10-5mol·L-1 for both of o-cresyl acetate and m-cresyl acetate,and in the range of (0.03-2.25)×10-5mol·L-1 for p-cresyl acetate,with their values of detection limit (3S/N) of 1.88×10-5mol·L-1 (for o- and m-cresyl acetate) and 1.13×10-5mol·L-1 (for p-cresyl acetate).Recovery of the method was tested by standard addition method using samples of o-,m- and p-cresol as matrixes,values of average recovery found were 104%,95% and 100% with values of RSD (n=7) of 0.22%,2.34% and 0.90% respectively.