Semi-automated quantification of hair cells in the mature mouse utricle

•Quantification of hair cell density by counting a subset of hair cells (HCs) in regions of interest (ROIs) reduces the accuracy of HC counts and increases the variability compared to counting all HCs in the utricle.•Manually counting HCs from the entire utricle is time-consuming and labor-intensive.•Pou4f3 immunolabeling results in a highly specific and discrete nuclear signal that allows semi-automated quantification of HCs using an ImageJ macro.•The semi-automated method provides accurate HC counts while significantly reducing the time required to quantify HCs in the entire utricle.•It is essential to confirm HC viability using a secondary method (such as activated caspase 3/7 labeling), since dead HCs can retain both Myosin7a and Pou4f3 immunoreactivity, especially in cisplatin-treated tissue.