This article describes reproducibility of a single-step automated β-galactosidase, and the equivalence of its data to the traditional assay ("Experiments in Molecular Genetics" [1]). This was done via a pairwise comparison of both methods using strains with Miller Unit [MU] values ranging from 0 to over 2000. The data presented in this article is associated with the research article entitled "A single-step method for mid to high throughput β-galactosidase assays in Escherichia coli using a microplate reader" [2].