Key message <p>TubeTracker provides a method to partially automate analysis of pollen tube growth using live imaging.</p> Abstract <p>Pollen function is critical for successful plant reproduction and crop productivity and it is important to develop accessible methods to quantitatively analyze pollen performance to enhance reproductive resilience. Here we introduce TubeTracker as a method to quantify key parameters of pollen performance such as, time to pollen grain germination, pollen tube tip velocity and maintenance of pollen tube integrity. TubeTracker integrates manual and automatic image processing routines and the graphical interface allows the user to interact with the software to make manual corrections of automated steps. TubeTracker does not depend on training data sets required to implement machine learning approaches and thus can be immediately implemented using readily available imaging systems. Furthermore, TubeTracker is an excellent tool to produce the pollen performance data sets necessary to take advantage of emerging AI-based methods to fully automate analysis. We tested TubeTracker and found it to be accurate in measuring pollen tube germination and pollen tube tip elongation across multiple cultivars of tomato.</p> Graphical abstract <p>Graphical user interface of TubeTracker showing all supported functionalities.</p>