Deoxyribozymes are synthetic and single stranded DNAs that are capable of catalysis of a variety of reactions, including cleavage of DNA substrates. Deoxyribozymes are usually characterized by analytical single-turnover kinetic assays, however, for many applications e.g. characterization of the reaction products, semi-preparative and preparative reactions are required. At such scales, there is a lack of comprehensive analysis and conditions that supports high amount of products in an appropriate time-scale are vaguely guessed by researchers. In this report, catalytic activity of an oxidizing DNA-cleaving deoxyribozyme, F-8(X), was comprehensively inspected in semi-preparative (10 μM substrate) scale. A 60 nucleotides long synthetic DNA sequence was selected as the target DNA for this study. The DNA sequence was originated from a single stranded DNA virus. Investigations revealed high yield in the presence of optimal concentration of oxidizing agents. The optimal conditions have been applied for scale-up of the reaction to preparative (40 μM substrate) and multi-turnover reactions to achieve highest amount of product in a cost-, time- and labor-effective manner. Such a comprehensive analysis of a deoxyribozyme's activity in semi-preparative scale provides a platform for expanded applications of DNA catalysts as a tool in chemical biology.