A novel procedure (CROSS: Computational Rescaffolding and Optimization using Synthetic Schemes) for in silico rescaffolding and side-chain optimization is reported with explicit consideration of the route of synthesis and availability of compatible chemical reagents. We have defined a set of retrosynthetic disconnections representing robust reactions, amenable for combinatorial chemistry. This rule set is used to generate virtual fragment databases from available reagents. Each reactive center is annotated with its compatibility with regard to the chemical reactions. The rule set is then applied to a new molecule to obtain separate query subunits for rescaffolding by 3D similarity searching in specific reagent-derived fragment databases. Thus, only fragments compatible with the chemistry and shape of the corresponding query moiety are investigated further. The identified fragment hits directly indicate (1) available chemical reagents that can replace the query moiety in the starting molecule and (2) the route for the synthesis of the proposed molecules.