Lattice light sheet microscopy is revolutionizing cell biology since it enables fast, high-resolution extended imaging in three dimensions combined with a drastic reduction in photo-toxicity and bleaching. However analysis of such data sets still remains a major challenge. Automated tracking of kinetochores, the protein complex facilitating and controlling microtubule attachment of the chromosomes within the mitotic spindle, provides quantitative assessment of chromosome dynamics in mitosis. Here we extend existing open-source kinetochore tracking software (KiT Armond et al. ) to track (and pair) kinetochores throughout pro-metaphase to anaphase in lattice light sheet microscopy data. One of the key improvements is a regularization term in the objective function to enforce biological information about the number of kinetochores in a human mitotic cell, as well as improved diagnostic tools. This software provides quantitative insights into how kinetochores robustly ensure congression and segregation of chromosomes during mitosis. KiT is free, open-source software implemented in MATLAB and can be downloaded as a package from https://github.com/cmcb-warwick/KiT. The source repository is available at https://bitbucket.org/jarmond/kit (tag v2.4.0) and under continuing development. Supplementary data are available at Bioinformatics online. © The Author(s) 2022. Published by Oxford University Press.