Objective: We aimed to validate a software, JLK LVO, that automatically detects large vessel occlusion (LVO) on computed tomography angiography (CTA) using deep learning, within a prospective multicenter dataset. In addition, we calibrated the predicted probability of LVO against observed frequency and assessed the clinical implications of LVO probability in terms of follow up infarct volume and functional outcome. Method: From 2021 to 2023, we prospectively collected data from patients who underwent CTA within 24 hours of symptom onset at six university hospitals in Korea. The diagnostic performance of the software was evaluated using the area under the curve (AUC), sensitivity, and specificity across the entire study population and specifically in patients with isolated middle cerebral artery (MCA) M2 occlusion. In addition, we compared LVO probabilities after stratifying patient into acute LVO, chronic LVO, isolated MCA M2 occlusion, relevant MCA stenosis, and without stenoocclusion of MCA groups. We calibrated LVO probabilities in two ways: through mathematical calibration using logistic regression, and by refining LVO probabilities based on the observed frequency of LVO. We then assessed the association of LVO probability categories with infarct volume on follow up diffusion weighted imaging (DWI) and modified Rankin Scale (mRS) scores three months poststroke, using ANOVA and the Cochran Armitage test. Results: After excluding 168 patients, 796 remained; the mean (SD) age was 68.9 (13.7) years, and 57.7% were men. LVO was present in 193 (24.3%) of these patients, and the median interval from last known well to CTA was 5.7 hours (IQR 2.5 to 12.1 hours). At default threshold of 0.5, the software achieved an AUC of 0.944 (95% CI 0.926 to 0.960), with a sensitivity of 0.896 (0.845 to 0.936) and a specificity of 0.904 (0.877 to 0.926). In isolated MCA M2 occlusion, the AUROC was 0.880 (95% CI 0.824 to 0.921). Compared to the without stenoocclusion of MCA groups (median LVO probability 0.5, interquartile range 0.1 to 6.5), relevant stenosis (median 15.3, 2.4 to 77.4) and isolated MCA M2 occlusion (82.1, 40.9 to 98.2) groups had significantly higher LVO probability. Due to sparse data between 20 to 60% of LVO probabilities, recategorization into unlikely (0 to 20% LVO scores), less likely (20 to 60%), possible (60 to 90%), and suggestive (90 to 100%) provided a reliable estimation of LVO compared with mathematical calibration. The category of LVO probabilities was significantly associated with follow up infarct volumes on DWI and 3 months mRS scores. Conclusion: In this multicenter validation study, we proved the clinical efficacy of the software in detecting LVO on CTA. Additionally, using large scale real world data, we calibrated the LVO probabilities, which may provide a more confident estimation of LVO for practicing physicians.