After his PhD, Wüthrich continued postdoctoral research with Fallab for a short time before leaving to work at the University of California, Berkeley for two years from 1965 with Robert E. Connick.  That was followed by a stint working with Robert G. Shulman at the Bell Telephone Laboratories in Murray Hill, New Jersey from 1967 to 1969.
Wüthrich returned to Switzerland, to Zürich, in 1969, where he began his career there at the ETH Zürich, rising to Professor of Biophysics by 1980. He currently maintains a laboratory at the ETH Zürich, at The Scripps Research Institute, in La Jolla, California and at the  of ShanghaiTech University. He has also been a visiting professor at the University of Edinburgh (1997–2000), the Chinese University of Hong Kong (where he was an Honorary Professor) and Yonsei University.
During his graduate studies Wüthrich started out working with electron paramagnetic resonance spectroscopy, and the subject of his PhD thesis was "the catalytic activity of copper compounds in autoxidation reactions". During his time as a postdoc in Berkeley he began working with the newly developed and related technique of nuclear magnetic resonance spectroscopy to study the hydration of metal complexes. When Wüthrich joined the Bell Labs, he was put in charge of one of the first superconducting NMR spectrometers, and started studying the structure and dynamics of proteins. He has pursued this line of research ever since.
After returning to Switzerland, Wüthrich collaborated with, among others, Nobel laureate Richard R. Ernst on developing the first two-dimensional NMR experiments, and established the nuclear Overhauser effect as a convenient way of measuring distances within proteins. This research later led to the complete assignment of resonances for among others the bovine pancreatic trypsin inhibitor and glucagon.
In October 2010, Wüthrich participated in the USA Science and Engineering Festivals Lunch with a Laureate program where middle and high school students will get to engage in an informal conversation with a Nobel Prize–winning scientist over a brown-bag lunch. Wüthrich is also a member on the USA Science and Engineering Festivals Advisory Board and a supporter of the Campaign for the Establishment of a United Nations Parliamentary Assembly, an organisation which campaigns for democratic reform in the United Nations.