<p>Mass spectrometry-based targeted quantitation technologies are widely recognized for their high sensitivity, strong specificity, broad dynamic range, and excellent quantitative accuracy, and have been established as a cornerstone technique for discovery, validation, and translation research in the proteomics field. Ongoing advancements in mass spectrometry hardware and software have significantly enhanced targeted proteomics analysis, enabling its application in a wide range of scientific disciplines. However, several technical and methodological challenges remain. Real-time acquisition strategies and algorithms have been developed with increased efficiency and intelligence. These strategies, namely, employing stable isotope triggering, real-time search localization, or retention time (RT) adjustment, have transformed targeted proteomics from a static analytical model into a dynamic and intelligent acquisition framework. This transition enables the detection of more targets per run while enhancing sensitivity, specificity, and quantification accuracy. This review focuses on novel strategies and technologies in mass spectrometry-based targeted proteomics. It provides a systematic description of the underlying principles, applications, and technical challenges of these strategies and technologies, with the intention to provide a comprehensive resource for an in-depth understanding of targeted quantitative proteomics.</p>