An HPLC method for the determination of 2,4-D in tomatoes has been studied. Tomato samples were extracted with acetone-dichloromethane (2:1) after hydrolysis. The extract was cleaned up with acid-base partition and furthermore eluting through XAD-2 column. Then, it was analyzed using reverse phase HPLC. Four different mobile phase mixtures, at two different flow rates of 1.0 and 1.5 mL/min, were tried, to choose the best conditions in the final HPLC determination step. The best results were obtained using the mixture of acetonitrile:water, containing 2% of acetic acid (50:50, v/v), at a flow rate of 1.0 mL/min for the separation of 2,4-D standard solutions, but the baseline separation of 2,4-D in the final solution of sample extract could not be achieved due to interferences causing poor resolution as well as low recovery.