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Posttranslational modification (PTM) of proteins is a versatile cellular process to regulate the activities of proteins. The high regioselectivity and catalysis rate of posttranslationally modifying enzymes utilizing high-energy precursors can potentially be exploited to equip proteins or peptide sequences with a label of choice site selectively and efficiently. We and others have recently described and analyzed a new reversible PTM called phosphocholination in which a phosphocholine group is transferred from a cytidine diphosphate choline (CDP-choline) to a serine residue of the small GTPase Rab1 [1–3]. The enzymes AnkX and Lem3 catalyze the modification and the corresponding demodification reactions, respectively. Interestingly, we could demonstrate that the modifying enzyme AnkX only requires a short amino acid sequence for substrate recognition. Therefore, we envision AnkX as a tool for the site directed labeling of target proteins. Here we report on the progress of developing a novel reversible protein labeling strategy based on the enzymes AnkX and Lem3 and on derivatives of CDP-choline. We demonstrate the optimization of AnkX and Lem3 enzyme activities and the identification of optimal and minimal peptide target sequences. Results indicate that indeed arbitrary proteins of interest can be functionalized with phosphocholine derivatives. In summary, this work yields first insights into the development of a CDP-choline based fully reversible protein labeling strategy.