© 2019, Springer Science+Business Media, LLC, part of Springer Nature.Brassinosteroids (BRs) are a class of polyhydroxysteroids that have been recognized as a “sixth class of plant hormones.” Exogenous application of BRs affects a broad spectrum of physiological responses, secondary metabolite accumulation, and resistance against stress factors in plants. In this study, the effects of pre-harvest 24-epibrassinolide (24-eBL) applications on the accumulation of antioxidant compound, yield, and quality properties of Alphonse Lavallée grape cultivar were examined. 24-eBL was applied to vines with 13 combinations including different application times and concentrations of 24-eBL (0.2, 0.4, 0.6, and 0.8 mg L−1) and control. It was found that 24-eBL increased yield, quality, and antioxidant compounds in grapes when it was applied at appropriate concentration and application time. The concentration 0.2 mg L−1 of 24-eBL applied to vines at three times (7 days after berry set + veraison + 30 days after veraison) was the most suitable application providing the highest yield and some quality properties such as cluster weight, berry weight, and specific gravity. Concentrations 0.6 and 0.8 mg L−1 of 24-eBL applied at veraison to vines can be suggested for total phenolics, β-carotene, ascorbic acid, and trans-resveratrol accumulation for Alphonse Lavallée as a practical application, while the application consists of 0.6 mg L−1 of 24-eBL and 7 days after berry set + veraison + 30 days after veraison seems to be the best treatment for anthocyanin content.