As a professor of pathology he taught from 1899 to 1903 at the University of Pennsylvania. Beginning in 1901, he first took over in addition to his teaching activities, the management of the newly established Rockefeller Institute for Medical Research and later sat on the Board of the Rockefeller Foundation.
During the severe polio epidemics in the early 20th century, he was regarded by his contemporaries as the scientist who could significantly advance the polio research. In 1909, Karl Landsteiner and Erwin Popper managed to infect monkeys with the polio virus, and they described a neurotropic, filtrable virus. Simon Flexner confirmed their findings. Flexner was able also to transmit the infection from a monkey to the next. This made it possible to study the disease in laboratory animals. Simon Flexner used for his studies, however, rhesus monkeys, which do not take one of the few species of primates, the virus via the mouth. While Simon Flexner achieved substantial progress in the treatment of meningitis, drew his results researching the polio disease for a long time on the wrong lines.
In 1912, the Princeton University Flexner awarded an honorary doctorate.
He was the brother of Abraham Flexner.