The work performed in Dr. Rhoton's Lab, represented by over 500 publications on microneurosurgical anatomy, greatly contributed to improving the level of neurosurgical treatment throughout the world. The authors reviewed the development and activities of the Lab over 40 years. Dr. Albert L. Rhoton Jr., the founder of, and leader in, this field, displayed great creativity and ingenuity during his life. He devoted himself to perfecting his study methodology, employing high-definition photos and slides to enhance the quality of his published papers. He dedicated his life to the education of neurosurgeons. His "lab team," which included microneuroanatomy research fellows, medical illustrators, lab directors, and secretaries, worked together under his leadership to develop the methods and techniques of anatomical study to complete over 160 microneurosurgical anatomy projects. The medical illustrators adapted computer technologies and integrated art and science in the field of microneurosurgical anatomy. Dr. Rhoton's fellows established methods of injecting colors and pursued a series of projects to innovate surgical approaches and instruments over a 40-year period. They also continued to help Dr. Rhoton to conduct international educational activities after returning to their home countries. Rhoton's Lab became a world-renowned anatomical lab as well as a microsurgical training center and generated the knowledge necessary to perform accurate, gentle, and safe surgery for the sake of patients.