The article reviews the recent developments in mass spectrometry-based targeted proteomics for clinical cancer biomarker analysis. It highlights the importance of routine measurement of cancer biomarkers in early detection, risk classification, and treatment monitoring, emphasizing the need for clinically validated assays. The review discusses the advantages and challenges of targeted proteomics, including high selectivity, specificity, applicability to different sample matrices, multiplexing capabilities, sample efficiency, dynamic range, and high reproducibility. It also addresses the regulatory requirements for clinical implementation, such as method development, validation, and quality assurance. Recent advancements in automation, sample preparation, and software capabilities are discussed, along with the potential of immunoaffinity enrichment and multidimensional LC to improve throughput and sensitivity. The article concludes by outlining future directions, including the potential of targeted proteomics in multi-biomarker panels and precision medicine, while acknowledging the high technical proficiency required for LC–MS/MS handling.The article reviews the recent developments in mass spectrometry-based targeted proteomics for clinical cancer biomarker analysis. It highlights the importance of routine measurement of cancer biomarkers in early detection, risk classification, and treatment monitoring, emphasizing the need for clinically validated assays. The review discusses the advantages and challenges of targeted proteomics, including high selectivity, specificity, applicability to different sample matrices, multiplexing capabilities, sample efficiency, dynamic range, and high reproducibility. It also addresses the regulatory requirements for clinical implementation, such as method development, validation, and quality assurance. Recent advancements in automation, sample preparation, and software capabilities are discussed, along with the potential of immunoaffinity enrichment and multidimensional LC to improve throughput and sensitivity. The article concludes by outlining future directions, including the potential of targeted proteomics in multi-biomarker panels and precision medicine, while acknowledging the high technical proficiency required for LC–MS/MS handling.