Methods for determining regulon enrichment in gene expression signatures are disclosed herein. An example method can include obtaining a set of transcriptional targets of a regulon. The method can include obtaining a gene expression signature by comparing a gene expression profile of a test sample to gene expression profiles of a plurality of samples representing control phenotypes. The method can include calculating a regulon enrichment score for each regulon in the gene expression signature. The method can including determining whether a number of control samples in the control phenotypes is above a predetermined threshold to support evaluation of statistical significance using permutation analysis. The method can include, in response to determining that the number of control samples is above the predetermined threshold, calculating a significance value by comparing each regulon enrichment score to a null model.