Introduction Intraoperative identification of parathyroid tissue (PT) is critical. Differentiating PT from surrounding tissue is challenging and Frozen Section (gold-standard) is resource intensive. Aspartate aminotransferase-to-lactate dehydrogenase (AST/LDH) ratio of suspended tissue distinguished PT from surrounding tissue in a Japanese study. Our study seeks to determine if this is reproducible in Australia. Methods Patients undergoing thyroidectomy and/or parathyroidectomy between August 2021 and June 2023 with appropriately processed tissue yielding AST/LDH ratio were included. 1mm^3 of PT and apparently distinct tissue were minced, suspended, AST/LDH ratio obtained and compared with histopathology. Mann-Whitney U test was used to determine a difference in AST/LDH ratio between parathyroid and non-parathyroid tissue. ROC curves were plotted and maximum Youden index calculated to determine optimal cut-off value with highest sensitivity and specificity. Results 68 specimens from 48 patients were included. 25 (36.76%) were histologically confirmed PT, 30 (44.12%), thyroid, 11 (16.18%), adipose tissue and 1 (1.47%) thymus. AST/LDH ratio in suspended PT specimens were higher than other tissues. The Mann-Whitney U test demonstrated a significant difference between parathyroid and non-parathyroid (p-value < 0.001). The area under the ROC curve (AUC) was 0.978. 0.157 was the optimal cut-off value with 96.15% sensitivity and 97.67% specificity. Conclusion We demonstrated that ASL/LDH ratio distinguishes PT from other tissues. High AUC indicates a highly sensitive and specific test. Optimal cut-off value was 0.157. This method may be a useful in resource-poor services and may increase access to PT surgery.