Secondary ion mass spectrometry (SIMS) has become an increasingly utilized tool in biologically relevant studies. Of these, high lateral resolution methodologies using the NanoSIMS 50/50L have been especially powerful within many biological fields over the past decade. Here, the authors provide a review of this technology, sample preparation and analysis considerations, examples of recent biological studies, data analyses, and current outlooks. Specifically, the authors offer an overview of SIMS and development of the NanoSIMS. The authors describe the major experimental factors that should be considered prior to NanoSIMS analysis and then provide information on best practices for data analysis and image generation, which includes an in-depth discussion of appropriate colormaps. Additionally, the authors provide an open-source method for data representation that allows simultaneous visualization of secondary electron and ion information within a single image. Finally, the authors present a perspective on the future of this technology and where they think it will have the greatest impact in near future.