Rapid and affordable detection of cancer specific targets, such as allele-specific single-nucleotide mutational changes, are of great need to improve treatment efficacy and guide clinical use. Detecting these mutations in isothermal conditions creates the opportunity to develop cost-efficient diagnostic platforms for cancer treatment. In this paper, a novel python script was constructed to develop a simple and personalised software tool that could automate the design of allele-specific primers in isothermal conditions using design parameters, such as free energies and annealing temperatures. The scripts’ primers were compared to manually designed probes that were experimentally tested on a variant (ESR1 p.E380Q), commonly present in metastatic breast cancer, showcasing the applicability of the automated software to design allele-specific assays for Lab-on-Chip platforms in cancer diagnostics.