Today, there is an increasing interest in antioxidants, especially to prevent the known harmful effects of free radicals in human metabolism and their deterioration during processing and storage of fatty foods. In both cases, natural-source antioxidants are preferred over synthetic antioxidants. So, there has been a parallel increase in the use of assays to estimate antioxidant efficacy in human metabolism and food systems. Today, there are many bioanalytical methods that measure the antioxidant effect. Of these, the 1,1-diphenyl-2-picrylhydrazil (DPPH) removing assay is the most putative, popular, and commonly used method to determine antioxidant ability. In this review, a general approach to the DPPH radical scavenging assay has been taken. In this context, many studies, including attempts to adapt the DPPH radical scavenging method to different analytes, search for the highest antioxidant activity values, and optimize the method of measurement, have previously been performed. Therefore, it is highly important to introduce measures aimed at standardizing the conditions of the DPPH radical scavenging activity, including the various reaction media suitable for this assay. For this aim, the chemical and basic principles of DPPH free radical scavenging are defined and discussed in an outline. In addition, this study describes and defines the basic sections of DPPH free radical scavenging in food and biological systems. Additionally, some chemical, critical, and technical details of the DPPH free radical removal method are given. This is a simple assay in which the prospective compounds or herbal extracts are mixed with the DPPH solution and their absorbance is measured after a certain period. However, despite rapid advances in instrumental techniques and analysis, this method has not undergone extreme modification. This study presents detailed information about the DPPH method and an in-depth review of different developments.