A number of theoretical and experimental approaches to design biologically active antisense oligonucleotides (AS-ON) have proven their usefulness. This includes systematic computational strategies that are based on the understanding of antisense mechanisms. Here, we investigate in detail the relationship between computational parameters of the local target search for the theoretical design of AS-ON and the hit rate, that is, the biologic efficacy of AS-ON in cell culture. The computational design of AS-ON studied in this work is based on an established algorithm to predict structurally favorable local target sites along a given target RNA against which AS-ON are directed. Briefly, a sequence segment of a certain length (window) is used to predict a group of lowest-energy RNA secondary structures. Subsequently, this window is shifted along the target sequence by a certain step width. To date, those technical parameters of the systematic structural target analysis have been chosen arbitrarily. Here, we investigate their role for the successful design of AS-ON and suggest an optimized computer-based protocol for the selection of favorable local target sequences and, hence, an improved design of active AS-ON. Further, this study provides systematic insights into the structure-function relationship of AS-ON.