The mechanisms by which genetic risk variants interact with each other, as well as environmental factors, to contribute to complex genetic disorders remain unclear. We describe in detail our recently published approach to resolve distinct additive and synergistic transcriptomic effects after combinatorial manipulation of genetic variants and/or chemical perturbagens. Although first developed for CRISPR-based perturbation studies of isogenic human induced pluripotent stem cell-derived neurons, our methodology can be broadly applied to any RNA sequencing dataset, provided that raw read counts are available. Whereas other differential expression analyses reveal the effect of individual perturbations, here we specifically query interactions between two or more perturbagens, resolving the extent of non-additive (synergistic) interactions between perturbations. We discuss the careful experimental design required to resolve synergistic effects and considerations of statistical power and how to quantify observed synergy between experiments. Additionally, we speculate on potential future applications and explore the obvious limitations of this approach. Overall, by interrogating the effect of independent factors, alone and in combination, our analytic framework and experimental design facilitate the discovery of convergence and synergy downstream of gene and/or treatment perturbations hypothesized to contribute to complex diseases. We think that this protocol can be successfully applied by any scientist with bioinformatic skills and basic proficiency in the R programming language. Our computational pipeline (https://github.com/nadschro/synergy-analysis) is straightforward, does not require supercomputing support and can be conducted in a single day upon completion of RNA sequencing experiments.