Solid by-products generated in the winemaking process, can comprise valuable bioactive substances such as resveratrol and viniferin, which can be used in whole range of sectors including medicine, pharmacy, cosmetic industry etc. The changes in content of those stilbenes in extracts obtained by maceration and Soxhlet extraction were monitored using newly modified and validated high-performance liquid chromatography-mass spectrometry method which was proved to be accurate, reproducible, and efficient for their determination. The yields of individual bioactive compounds isolated from winery by-products are crucially dependent on the conditions of used extraction techniques. From this point of view, stability testing including light exposure, elevated temperature, and storage for longer time periods in the solution, represents the basis for optimizing conditions of extraction methods of resveratrol and trans-ε-viniferin. High temperature is beneficial for better release of thermally more stable stilbenes such as trans-resveratrol and trans-ε-viniferin but its application for prolonged time periods can be destructive. Light stress conditions cause the formation of otherwise unavailable cis-ε-viniferin by dimerization and photoisomerization of trans- stilbenes.