This research utilized a real raw diesel oil to be subjected in an innovative desulfurization technique. The novelty of this study is in the use of screening analysis in the ultrasonication enhancement of sulfur oxidation in actual fuel. This addresses the research gap in literature that have yet to compare and completely analyze all factors in the ultrasound-assisted oxidative desulfurization (UAOD) method. Specifically, the effects of ultrasonication via amplification and irradiation time, material usage of tetraoctylammonium bromide, hydrogen peroxide, polyoxometalate catalyst and the process parameters of fuel to oxidant molar ratio and temperature were examined. A definitive screening design (DSD) using the JMP 11.0 was utilized for a statistical screening analysis to determine the essential and non-essential parameters in the UAOD. Upon the oxidation of sulfur compounds in diesel oil, results suggested that the enhancement technique via ultrasonication, polyoxometalate catalyst and temperature were the only significant factors based on the p-value < 0.05. The aforementioned factors are essential due to being able to instigate an oxidation reaction of sulfurs into its sulfone forms. Based on the generated DSD experimental runs, sulfur conversion can range from 36.41 % to 92.69 %.