Together with Barry Sharpless and M.G. Finn, Kolb developed the concept of click chemistry, an approach to simplify synthesis by focusing on a few chemical reactions that are similar in nature. The associated scientific publication Click chemistry: diverse chemical function from a few good reactions has been cited more than 14,000 times (as of 2021) and was awarded the 2022 Nobel Prize in Chemistry for K. Barry Sharpless, Carolyn Bertozzi and Morten Meldal. Kolb refined the method by combining it as in-situ click chemistry with microfluidic processes. This makes it particularly easy to synthesize new inhibitors for various enzymes. Kolb's more recent work deals with the synthesis of new tracers for positron emission tomography (e.g. for detecting the tau protein in Alzheimer's disease) and with the clinical testing of these tracers, a key highlight being [18F]-T807, also known as AV1451, Flortaucipir, Tauvid, which was approved in 2020 by the US food and drug administration (FDA) for imaging neurofibrillary tangles in adults who are being evaluated for Alzheimer’s Disease.

Kolb’s lab has developed a blood plasma assay for phospho-217-Tau (p217Tau), which shows potential as a highly accurate peripheral biomarker for amyloid and Tau status in Alzheimer’s Disease.