Protein lysine acetylation (Kac), including histone acetylation and non-nuclear protein acetylation, is a dynamic and reversible post-translational modification for cellular regulation. The modified proteins play a key role in regulating chromatin structure, transcriptional activity and metabolic pathways, thus contributing to diverse cellular processes like transcription, cell cycle regulation, apoptosis and senescence. Therefore, targeting protein acetylation represents a potentially promising strategy for certain diseases, such as cancer. However, global identification of protein acetylation is a major bottleneck due to its dynamic property and rather low abundance. Tremendous efforts have been made to develop mass spectrometry (MS)-based proteomic technologies for this purpose from diverse cellular sources. The present review has tried to provide an overview of current strategies employed for Kac identification from histone to system-wide Kac analysis, including enrichment techniques, chromatographic separation strategies, and mass spectrometry methods.