TrackMate is an open-source Fiji plugin designed for automated, semi-automated, and manual tracking of single particles. It offers a versatile and modular solution with a user-friendly interface, making it accessible to researchers. TrackMate supports various imaging modalities, including 1D, 2D, and 3D over time, and can handle single and multi-channel image variants. The software includes visualization tools and data export capabilities, allowing for integration with other analysis tools. TrackMate's data model is flexible, enabling the detection and linking of particles, and its modular design allows for easy customization and extension by developers. The article presents three biological applications of TrackMate: lineage analysis in *Caenorhabditis elegans* to assess the impact of light-induced damage on early development, investigation of NEMO (NF-κB essential modulator) clusters in fibroblasts to study the effects of phototoxicity on motility analysis, and quantitative lifetime analysis of clathrin-mediated endocytosis in plant cells. These applications demonstrate TrackMate's versatility and accuracy in handling complex biological processes.TrackMate is an open-source Fiji plugin designed for automated, semi-automated, and manual tracking of single particles. It offers a versatile and modular solution with a user-friendly interface, making it accessible to researchers. TrackMate supports various imaging modalities, including 1D, 2D, and 3D over time, and can handle single and multi-channel image variants. The software includes visualization tools and data export capabilities, allowing for integration with other analysis tools. TrackMate's data model is flexible, enabling the detection and linking of particles, and its modular design allows for easy customization and extension by developers. The article presents three biological applications of TrackMate: lineage analysis in *Caenorhabditis elegans* to assess the impact of light-induced damage on early development, investigation of NEMO (NF-κB essential modulator) clusters in fibroblasts to study the effects of phototoxicity on motility analysis, and quantitative lifetime analysis of clathrin-mediated endocytosis in plant cells. These applications demonstrate TrackMate's versatility and accuracy in handling complex biological processes.