Kinesin motors couple ATP hydrolysis to movement along microtubules, which act both as tracks and as activators of kinesin ATPase activity. Cryo-electron microscopy and image processing enables generation of three-dimensional snapshots of kinesin motors on their tracks at different stages of their ATPase cycle, and can reveal their motor mechanisms at secondary structure resolution. Here, we describe in detail the methods and conditions employed in our lab to prepare high-quality frozen-hydrated samples, which yield structural insights into kinesin motor mechanisms.