Most biomarker assays are typically designed to detect a single molecule type (DNA, RNA, proteins, etc.) in a single assay. This means that monitoring a diverse biomarker panel could quickly become a complex endeavor, requiring different techniques, labs, and expertise. In this chapter, we describe a method for multiplexed biomarker detection from a single sample using variable-length DNA payload chains as the output signal. Through the course of the assay, payloads are systematically disassembled in the presence of specific biomarkers. The resulting distinctly sized fragments then yield characteristic gel electrophoresis band patterns, which can be detected and quantified using image analysis algorithms. We detail the entire process for constructing DNA payloads and conducting a biomarker detection assay, including the sequence design, laboratory assembly, running the assay, and final image analysis.