Cryo-electron Microscopy is now a standard technique of choice along with protein crystallography to obtain atomic resolution of macromolecular assemblies, including viruses, either in apo form or complexed with ligands. Any cryo-EM project relies on optimized biochemistry and sample preparation methods including but not limited to plunge freezing or high pressure freezing of samples, potentially thinning biological samples using focused ion beam milling, or utilizing hybrid approaches like cryo-CLEM, micropatterning etc. Recently developed biochemical techniques to be used for preparing samples for cryo-EM include cell free protein expression (CPE) for soluble as well as membrane proteins. While several reports have been published on each of these approaches, it is not possible to include each and every detail of the sample preparation workflow in a word restricted manuscript. Moreover, watching a specialized sample preparation workflow performed by an experienced researcher can help researchers newly venturing into cryo-EM to optimize sample preparation specific for their research. We are also including methods including novel methods of protein or sample purification and grid preparation which lead to improvement in cryo-EM imaging over existing methods. The aim of this Methods Collection is to make available a broad collection of methods that are geared towards any form of cryo-EM sample preparation.