This review discusses the concept of deconvolution of multi-mutational variants in protein engineering, particularly in the context of directed evolution of stereoselective enzymes. It highlights how mutations can interact cooperatively or antagonistically, not just additively, and how this has been studied using molecular dynamics (MD) and quantum mechanics/molecular mechanics (QM/MM) computations. Data from complete deconvolution can construct unique multi-dimensional rugged fitness pathway landscapes, providing insights different from traditional fitness landscapes. The review also discusses the use of double mutant cycles to study mutational effects and the importance of deconvolution in understanding non-additive effects. It covers several case studies, including the deconvolution of a triple mutant lipase variant, a quadruple mutant Baeyer–Villiger monooxygenase variant, and a quintuple cyclohexyl amine oxidase variant. The review concludes with suggestions for future work and calls for a unified understanding of non-additivity and epistasis in protein engineering.This review discusses the concept of deconvolution of multi-mutational variants in protein engineering, particularly in the context of directed evolution of stereoselective enzymes. It highlights how mutations can interact cooperatively or antagonistically, not just additively, and how this has been studied using molecular dynamics (MD) and quantum mechanics/molecular mechanics (QM/MM) computations. Data from complete deconvolution can construct unique multi-dimensional rugged fitness pathway landscapes, providing insights different from traditional fitness landscapes. The review also discusses the use of double mutant cycles to study mutational effects and the importance of deconvolution in understanding non-additive effects. It covers several case studies, including the deconvolution of a triple mutant lipase variant, a quadruple mutant Baeyer–Villiger monooxygenase variant, and a quintuple cyclohexyl amine oxidase variant. The review concludes with suggestions for future work and calls for a unified understanding of non-additivity and epistasis in protein engineering.