Hairpin pyrrole‐imidazole polyamides (hPIPs) and their chlorambucil (Chb) conjugates (hPIP‐Chbs) can alkylate DNA in a sequence‐specific manner, and have been studied as anticancer drugs. Here, we conjugated Chb to a cyclic PIP (cPIP), which is known to have a higher binding affinity than the corresponding hPIP, and investigated the DNA alkylation properties of the resulting cPIP‐Chb using the optimized capillary electrophoresis method and conventional HPLC product analysis. cPIP‐Chb conjugate 3 showed higher alkylation activity at its binding sites than did hPIP‐Chb conjugates 1 and 2. Subsequent HPLC analysis revealed that the alkylation site of conjugate 3, which was identified by capillary electrophoresis, was reliable and that conjugate 3 alkylates the N3 position of adenine as do hPIP‐Chbs. Moreover, conjugate 3 showed higher cytotoxicity against LNCaP prostate cancer cells than did conjugate 1 and cytotoxicity comparable to that of conjugate 2. These results suggest that cPIP‐Chbs could be novel DNA alkylating anticancer drugs.