Lysine acetyl transferases (KATs) and lysine deacetylases (KDACs) are versatile enzymes that regulate the acetylation state of histone and non-histone proteins. KDACs are overexpressed in various types of cancer, and the inhibition thereof results in differentiation and apoptosis of tumour cells. This effect has been investigated in numerous diseases ranging from blood malignancies and solid tumours to viral infections. To date, however, the greatest medical benefits were observed in the treatment of blood cancer. KDAC inhibitor (KDACi) treatment is FDA-approved only for T cell lymphoma and multiple myeloma as a single agent or as a combinatorial therapy, respectively. Nevertheless, recent research shows promising results in extending the application of KDACis beyond blood malignancies to cardiovascular diseases, various inflammatory and neurodegenerative disorders. This thesis focuses on several aspects to investigate how KDACis influence cancer-specific pathways, particularly proteins associated with cell proliferation. The systems-biology approach was utilised to investigate an early response to KDACis in order to focus on the initial pathways that are altered after KDACi treatment, without triggering a plethora of secondary apoptotic events. This enabled identification of the proteins that are altered in expression and acetylation after KDACi treatment. Moreover, correlation of altered gene and protein expression denoted KDACi-specific modes-of-action. Our results revealed that the energy metabolism and nucleotide synthesis-related pathways are affected even under mild, non-apoptotic drug treatment. These also included proteins that are essential for cell survival. In manuscript #1, the global data is debated. Additional discussion and the future perspectives of this thesis focus on cancer-specific metabolic pathways, and the potential of exploiting KDACi-mediated acetylation changes to examine novel therapeutic strategies. To this end, the influence of altered acetylation of MTHFD1 and SHMT2 was investigated in the context of protein structure, oligomerisation potential, and enzyme function. Modification of lysine residues by acetylation has been identified on both histone and non-histone proteins. The analysis of altered histone acetylation, however, requires adaptation of the mass spectrometry-based (MS) approaches compared to analysis of non-histone protein acetylation. This is mostly due to differences in protein chemistry, i.e., length and sequence of the modified peptides. To this end, our FASIL-MS approach was developed to quantitate histone site-specific lysine acetylation mediated by KDACi treatment. FASIL-MS combines a streamlined sample preparation protocol, adjustment of the MS method and an improvement in the bioinformatic tool used for MS2-based quantitation of histone acetylation. This can be further employed in various experimental settings, such as monitoring drug- or time-dependent alterations in histone acetylation. Moreover, a combination of FASIL-MS with chromatin immunoprecipitation sequencing (ChIP-seq) can be utilised to localise certain acetylation changes at specific gene promoters. Multiple experimental approaches are needed to systematically assess the role of KDACis. To our knowledge, this thesis comprises the most comprehensive strategy to investigate the quantitative changes after KDACi treatment in gene and protein expression, together with altered histone and non-histone acetylation. A combination of these methods with additional tools to explore the function of acetylation changes will undoubtedly shed new light on alternative strategies for targeted cancer treatment.