Updated project metadata. Protein AMPylation is a prevalent posttranslational modification with an emerging role in neurodevelopment and neurodegeneration. Although in metazoans the two highly conserved protein AMP-transferases together with diverse group of AMPylated proteins have been identified using chemical proteomics and biochemical techniques the function of this modification remains largely unknown. Particularly problematic is a localisation of thus far identified AMPylated proteins and putative AMP-transferases. Here, we uncover protein AMPylation as a novel lysosomal protein posttranslational modification characteristic for differentiating neurons. The AMPylated soluble form of exonuclease PLD3 localised in lysosomes shows dramatic increase during the differentiation of neuronal cell lineages. Similar AMPylation pattern has been observed for a lysosomal acid phosphatase ACP2. Our discovery was enabled by combination of chemical proteomics and novel gel-based separation technique of modified and non-modified proteins. Together, our findings expose further the connection between the protein AMPylation and neurodevelopment and reveal a novel lysosomal posttranslational modification.