Widefield fluorescence microscopy has long been an invaluable tool in biomedical research. More recently, application of this technique has further increased with the introduction of new fluorophores and significant advances in optical instrumentation. More specifically, this technique has been used in a broad range of biological studies involving, for example, colocalization analysis, ratiometric imaging, 3D single molecule tracking and multicolor super-resolution imaging. Advanced widefield microscopy setups are generally implemented with many optical components, including mirrors, dichroic filters, excitation/emission filters, beam splitters, objective lenses, and optical cameras. The complexity of such microscopy configurations imposes an inherent risk of optical aberrations and systematic errors which can affect the quality and analysis of the acquired image data. Many methods have been introduced over the years to characterize specific aspects of fluorescence microscopy such as the system’s point spread function and field illumination. However, methods for the assessment of the various optical components, such as the objective lens, optical filter, and other key components required for microscope imaging, are lacking, and in general, there is a shortage of software tools for this analysis. Therefore, we present here a comprehensive system calibration protocol for improving the entire experimental pipeline, starting with image acquisition and ending with automated data analysis and workflow documentation. The protocol details the characterization of optical components, the assessment of data quality and validity, and correction of aberrations to allow for the attainment of the performance limits of the imaging system. This protocol allows researchers ranging from novice microscopists to imaging professionals to implement an optimal widefield imaging system.