Non-coding variants can alter transcription factor (TF)-DNA binding and dysregulate gene expression. Here, we present a protocol for evaluating the impact of non-coding variants on TF binding and gene expression. We describe steps for expressing and purifying DNA-binding proteins, preparing DNA probes and reporter constructs, and measuring changes in TF-DNA binding affinity. Finally, we provide instructions for quantification and statistical analysis. For complete details on the use and execution of this protocol, please refer to Peña-Martínez and Messon-Bird et al.1.