Cancers are caused by accumulated DNA mutations. This recognition of the central role of mutations in cancer and recent advances in next-generation sequencing, has initiated the massive screening of clinical samples and the identification of 1000s of cancer-associated gene mutations. However, proteomic analysis of the expressed mutation products lags far behind genomic (transcriptomic) analysis. With comprehensive global proteomics analysis, only a small percentage of single nucleotide variants detected by DNA and RNA sequencing have been observed as single amino acid variants due to current technical limitations. Proteomic analysis of mutations is important with the potential to advance cancer biomarker development and the discovery of new therapeutic targets for more effective disease treatment. Targeted proteomics using selected reaction monitoring (also known as multiple reaction monitoring) and parallel reaction monitoring, has emerged as a powerful tool with significant advantages over global proteomics for analysis of protein mutations in terms of detection sensitivity, quantitation accuracy and overall practicality (e.g., reliable identification and the scale of quantification). Herein we review recent advances in the targeted proteomics technology for enhancing detection sensitivity and multiplexing capability and highlight its broad biomedical applications for analysis of protein mutations in human bodily fluids, tissues, and cell lines. Furthermore, we review recent applications of top-down proteomics for analysis of protein mutations. Unlike the commonly used bottom-up proteomics which requires digestion of proteins into peptides, top-down proteomics directly analyzes intact proteins for more precise characterization of mutation isoforms. Finally, general perspectives on the potential of achieving both high sensitivity and high sample throughput for large-scale targeted detection and quantification of important protein mutations are discussed.