The study was carried out to purify protease produced by Bacillus subtilis ATCC 6633 strain and to examine the effect of the crude saponin extract (CSE) obtained from Gypsophila pilulifera Boiss. & Heldr. on the purified enzyme. In addition, the present study dealt with the evaluation of antioxidant and DNA cleavage potentials of the extract. Protease from B. subtilis ATCC 6633 was produced and purified using ammonium sulfate precipitation, gel filtration chromatography with 27.2-fold. The purified enzyme showed a single band on SDS-PAGE, and it was determined that its molecular weight was 30.1 kDa. In addition, to examine the effect of saponin as natural surfactant on the protease activity, crude saponin from G. pilulifera was extracted in methanol and the purified protease was incubated with the extract. It was observed that the activity of enzyme increased in the presence of extract. In the in vitro assays, while total phenolic content of the extract was determined by Folin-Ciocalteau method, antioxidant properties were evaluated by DPPH (1,1-diphenyl-2-picryl-hydrazyl) free radical scavenging activity, FRAP (ferric-ion reducing antioxidant parameter), CUPRAC (cupric ion reducing antioxidant capacity) and beta-caroten-linoleic acid method. The results obtained in this study showed that the extract possesses low antioxidant activity for studied all in vitro models. Also, effect of the extract on plasmid DNA was determined with agarose gel electrophoresis. The extract caused conversion of supercoiled DNA structure to other forms. As a result, the extract exhibited DNA cleavage activity as dose-dependent.