Anthropogenic contaminants can exhibit adverse effects on exposed organisms, including genotoxicity, resembling DNA damage. A common method to evaluate genotoxicity in exposed organisms is the micronucleus (MN) assay. It provides an efficient assessment of chromosomal impairment due to either chromosomal rupture or mis-segregation during mitosis. However, evaluating chromosomal damage in the MN assay through manual microscopy is a highly time-consuming and somewhat subjective process. High-throughput evaluation with automated image-analysis could reduce subjectivity and increase accuracy and throughput. In this study, we optimised the HiTMiN assay, adapting the MN assay to a miniaturised, 96-well-plate format and applied it to primary cells from green turtle fibroblasts. Image-analysis improved precision and reduced time compared to manual scoring. The assay was validated through exposure to two inorganic (chromium, cobalt) and one organic (metolachlor) compound, which are genotoxicants of concern in the marine environment.. Once validated, genotoxicity of turtle blood extracts were assessed from captured turtles of three major foraging grounds within Port Curtis, Gladstone, Queensland. All sampling sites induced MN formation with spatial variability observed. The HiTMiN assay presented here greatly increases the suitability of the MN assay as a quick and accurate assay to measure genotoxicity of environmental samples in cultured cell lines.