The aim of this study is an evaluation of the interaction between deoxyribonucleic acid (DNA) and anticancer drug Temozolomide (TMZ) and its degradation product. Such interactions were detected by high-performance liquid chromatography with diode array detector (HPLC-DAD). TMZ is unstable at physiological pH and completely decomposes. Hence, incubations of TMZ and DNA were performed in pH 4.5 acetate buffer. A C18 column and 0.02 M pH 4.5 acetate buffer-acetonitrile (90:10, v/v) as a mobile phase were used. In the chromatograms, it was observed that the peak area values of DNA increased by adding TMZ. This shows that TMZ and DNA are interacting in intercalation mode. Obtained results were verified by absorption and fluorescence spectrometry methods. By using HPLC and spectrophotometric methods, binding constants were calculated as 3.7x 10(4 )M(-1)and 2.6x 10(4 )M(-1), respectively. Besides, the interaction of degradation product of TMZ and DNA was also investigated.