In this study on human Tousled-Like-Kinase 2 (TLK2) mass spectrometry-based analysis was applied as part of a molecular characterization of TLK2 in the aim to increase the understanding of the mode of activation of this protein. Specifically, different TLK2 constructs were analyzed to generate a “phosphorylation map” of the TLK2 protein. To analyse the phosphorylation state of TLK2 we used recombinant protein purified from Escherichia coli. Samples of different TLK2 constructs were used for in-gel digestion and analysed by mass spectrometry. All samples were prepared and run in triplicates for label-free quantification. Explanation for MS raw files with TLK2 construct included: Ac: TLK2 construct consisting of amino acid residues 191-772 (Ac indicates active form). KD: TLK2 construct consisting of amino acid residues 191-772 with mutation D613A (KD indicates kinase-dead form). FusionKD: Kinase-dead part of a heterodimeric TLK2 construct (amino acid residues 191-772) with mutation D613A (KD indicates kinase-dead form). FusionAC: Active part of a heterodimeric TLK2 construct (amino acid residues 191-772) (Ac indicates kinase-dead form). KDlong: Kinase domain of TLK2 with C-tail (amino acid residues 450-772). KDshort: Kinase domain of TLK2 without C-tail (amino acid residues 450-753).