Rhizosphere bacterial communities have become a major focal point of research in recent years, especially regarding how they affect plants and vice versa (Philippot et al., 2013). Changes in microbial density and diversity within the rhizosphere occur in a spatial temporal manner. The soil zone closest to the plant roots has the most density and diversity of microbes (Clark, 1940). The lack of methods to consistently isolate rhizosphere samples in a spatially defined manner is a major bottleneck in rhizosphere microbiology. We hypothesized that microbes with increasing affinities to and distance from the plant root can be isolated using increasing strengths of physical disruption. Sonication is an excellent choice due to the ability to gently remove rhizosphere soil and bacterial biofilms without damaging plant roots (Doi T et al., 2007; Bulgarelli et al., 2012; Lundberg et al., 2012). In addition, simply increasing the time of sonication can increase the amount of physical force. We used such an approach to consistently isolate microbial communities with different affinities to the soybean roots (White et al., 2014). This article describes the use of successive sonication to isolate distal, middle, and proximal soil from the rhizosphere of soybean roots.