While the Staudinger reaction has first been described a hundred years ago in 1919, the ligation reaction became one of the most important and efficient bioconjugation techniques in the 1990s and this century. It holds the crucial characteristics for bioorthogonal chemistry: biocompatibility, selectivity, and a rapid and high-yielding turnover for a wide variety of applications. In the past years, it has been used especially in chemical biology for peptide/protein synthesis, posttranslational modifications, and DNA labeling. Furthermore, it can be used for cell-surface engineering, development of microarrays, and drug delivery systems. However, it is also possible to use the reaction in synthetic chemistry for general formation of amide bonds. In this review, the three major types, traceless and nontraceless Staudinger Ligation as well as the Staudinger phosphite reaction, are described in detail. We will further illustrate each reaction mechanism and describe characteristic substrates, intermediates, and products. In addition, not only its advantages but also stereochemical aspects, scope, and limitations, in particular side reactions, are discussed. Finally, the method is compared to other bioorthogonal labeling methods.