Twenty-five years ago, GFP revolutionized the field of cell biology by enabling scientists to visualize, for the first time, proteins in living cells. However, when it comes to current, state-of-the-art imaging technologies, fluorescent proteins (such as GFP) have several limitations that result from their size and photophysics. Over the past decade, an elegant, alternative approach, which is based on the direct labeling of proteins with fluorescent dyes, has been introduced. In this approach, an unnatural amino acid that can covalently bind a fluorescent dye is incorporated into the coding sequence of a protein, using genetic code expansion approaches (GCE). The protein of interest is thereby site-specifically fluorescently labeled inside the cell, eliminating the need for protein- or peptide-labeling tags. In the current project we have developed this labeling approach for cell biology applications and have demonstrated its advantages for live cell imaging and super resolution microscopy. By doing so, we provide a superior labelling approach for proteins in cells with reduced size, phototoxicity and increased photostability. This approach offers an attractive, improved alternative to GFP, by enabling tracking proteins in live cell with improved spatiotemporal resolution.