In this series, Dr. Anne Carpenter and Dr. Kevin Eliceiri provide an overview of bioimage analysis. Pre-processing is the first step that follows image acquisition and will prepare your image by reducing the signal-to-noise ratio, applying appropriate filters to the image, and color extraction. Once you perform pre-processing, you’re ready for segmentation, the process of identifying individual cells or structures within an image. If appropriate for your dataset, you can use tracking to be able to link objects in space and time and measure speed, directionality, and cell division. The last step of bioimage analysis is to analyze the data by measuring different features like the number of cells or biological structures, or their size, shape, intensity or texture. Carpenter and Eliceiri finalize this series by providing tips on best practices that will aid scientists in properly analyzing their data.