Abstract Introduction. Development of new therapies for cancer and infections frequently involves combinations of two or more drugs. Existing methods to quantify drug interactivity are suitable for analyzing drugs with parallel concentration-effect curves and similar maximum drug effects. This study established a method for the remaining situations. Methods. Equations describing zero-interactivity effects of combination therapy were developed based on the principle that a drug cannot interact with itself (i.e., additivity only). These equations were used to convert the concentration-effect (C-E) curve of the combination to C-E curve for each of the two drugs in the combination as if they were used as single agents (single agent-equivalent curves). Confidence intervals (CI) of these curves were generated using the error propagation method. Effect data of drug combinations were analyzed by the new and existing methods. Results. The calculated single agent-equivalent curves plus 95% CI defined the boundaries of an envelope for additive drug effects (Uncertainty Envelope). The Envelope shape and size were determined by differences in the shapes of single agent C-E curves. The nature of drug interactivity is indicated by the location of experimental data points of the combination; data points located within the Envelope indicate additivity, whereas points above and below the Envelope respectively indicate statistically significant synergy and antagonism. In comparison with the existing methods, the Envelope method yielded more conservative results (additivity vs. synergy or antagonism). Conclusion. Uncertainty Envelope method enables the interactivity analysis between drugs with nonparallel C-E curves and unequal maximal effect, with statistical confidence. Citation Format: Liang Zhao, Jessie L.-s. Au, M. Guill Wientjes. Uncertainty envelope to evaluate drug interactivity with statistical analysis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3485. doi:10.1158/1538-7445.AM2015-3485