The increased interest in (enzymatic) transformations between nucleosides and nucleobases has demanded the development of efficient analytical tools. In this report, we present an update and extension of our recently described method for monitoring these reactions by spectral unmixing. The presented method uses differences in the UV absorption spectra of nucleosides and nucleobases after alkaline quenching to derive their ratio based on spectral shape by fitting normalized reference spectra. It is applicable to a broad compound spectrum comprising more than 35 examples, offers HPLC‐like accuracy, ease of handling and significant reductions in both cost and data acquisition time compared to other methods. This contribution details the principle of monitoring reactions by spectral unmixing, gives recommendations regarding solutions to common problems and applications that necessitate special sample treatment. We provide software, workflows and reference spectra that facilitate the straightforward and versatile application of the method.DFG, 390540038, EXC 2008: Unifying Systems in Catalysis "UniSysCat"TU Berlin, Open-Access-Mittel – 2020DFG, 392246628, Chemo-enzymatische Synthese von Selen-modifizierten Nukleosiden, Nukleotiden und Oligonukleotide
