Single-ion mass spectrometry (SI-MS) enables accurate mass measurements for heterogeneous and high molecular weight samples, overcoming limitations of conventional mass spectrometry (MS). Charge detection mass spectrometry (CD-MS) and Orbitrap individual ion mass spectrometry (I²MS) are key techniques that allow precise determination of individual ion masses. CD-MS uses electrostatic induction to detect ions, while I²MS employs a commercial platform for high-resolution measurements. These methods are crucial for analyzing complex samples such as viruses, gene therapies, and vaccines, which are challenging for conventional MS due to their heterogeneity. CD-MS provides high-resolution mass distributions, enabling the identification of complex mixtures and subpopulations. The precision of SI-MS is determined by the root-mean-square deviation (RMSD) of measurements, with improvements in charge and m/z resolution enhancing the resolving power. CD-MS and I²MS have been applied to study various samples, including recombinant adeno-associated virus (rAAV) gene therapy vectors and adenovirus, revealing detailed information on their mass distributions and structural characteristics. The application of SI-MS extends to polymers, water droplets, and other macromolecules, demonstrating its versatility. Future developments aim to enhance resolution and throughput, making SI-MS a powerful tool for analyzing high-mass and complex samples.Single-ion mass spectrometry (SI-MS) enables accurate mass measurements for heterogeneous and high molecular weight samples, overcoming limitations of conventional mass spectrometry (MS). Charge detection mass spectrometry (CD-MS) and Orbitrap individual ion mass spectrometry (I²MS) are key techniques that allow precise determination of individual ion masses. CD-MS uses electrostatic induction to detect ions, while I²MS employs a commercial platform for high-resolution measurements. These methods are crucial for analyzing complex samples such as viruses, gene therapies, and vaccines, which are challenging for conventional MS due to their heterogeneity. CD-MS provides high-resolution mass distributions, enabling the identification of complex mixtures and subpopulations. The precision of SI-MS is determined by the root-mean-square deviation (RMSD) of measurements, with improvements in charge and m/z resolution enhancing the resolving power. CD-MS and I²MS have been applied to study various samples, including recombinant adeno-associated virus (rAAV) gene therapy vectors and adenovirus, revealing detailed information on their mass distributions and structural characteristics. The application of SI-MS extends to polymers, water droplets, and other macromolecules, demonstrating its versatility. Future developments aim to enhance resolution and throughput, making SI-MS a powerful tool for analyzing high-mass and complex samples.