Acetylation is a widely occurring post-translational modification (PTM) of proteins that plays a crucial role in many cellular physiological and pathological processes. Over the last decade, acetylation analyses required the development of multiple methods to target individual acetylated proteins, as well as to cover a broader description of acetylated proteins that comprise the acetylome. Areas covered: This review discusses the different types of acetylation (N-ter/K-/O-acetylation) and then describes some major strategies that have been reported in the literature to detect, enrich, identify and quantify protein acetylation. The review highlights the advantages and limitations of these strategies, to guide researchers in designing their experimental investigations and analysis of protein acetylation. Finally, this review highlights the main applications of acetylomics (proteomics based on mass spectrometry) for understanding physiological and pathological conditions. Expert opinion: Recent advances in acetylomics have enhanced knowledge of the biological and pathological roles of protein acetylation and the acetylome. Besides, radiolabeling and western blotting remain also techniques-of-choice for targeted protein acetylation. Future challenges in acetylomics to analyze the N-ter and K-acetylome will most likely require enrichment/fractionation, MS instrumentation and bioinformatics. Challenges also remain to identify the potential biological roles of O-acetylation and cross-talk with other PTMs.