Although the reaction between an azide and a phosphane to form an aza-ylide was discovered by Hermann Staudinger more than 80 years ago and has found widespread application in organic synthesis, its potential as a highly chemoselective ligation method for the preparation of bioconjugates has been recognized only recently. As the two reaction partners are bioorthogonal to almost all functionalities that exist in biological systems and react at room temperature in an aqueous environment, the Staudinger ligation has even found application in the complex environment of living cells. Herein we describe the current state of knowledge on this reaction and its application both for the preparation of bioconjugates and as a ligation method in chemical biology.