Abstract Diffuse large B-cell lymphoma (DLBCL) is the most common and aggressive lymphoma. Germinal center B-cell (GCB) and activated B-cell (ABC) are the two major biologically distinct molecular subtypes of DLBCL. Differentiation of these subtypes is essential for the right therapy. Gene expression signatures analysis for diagnosis or monitoring or predicting recurrence of various cancers is widely used in clinical practice. However, current assays require multiple qPCR reactions to analyze the gene signatures, increasing the health care cost burden. Multiplexing qPCR technologies that can detect multiple genes are of high clinical significance. To meet this need, this SBIR Phase I application proposes to develop a single RT-qPCR reaction Zip-MeltTM DLBCL Classifier Assay, an 18 gene expression analysis, to differentiate ABC and GCB subtypes. Our high multiplex assay has advantages as it can be done on a small amount of precious clinical samples, will reduce the turnaround time, and labor cost and ultimately will decrease the health care cost. The competitive advantage of our multiplex technology is that it expands the capacity of qPCR systems, can be easily implemented on any existing qPCR systems, reduces the operational cost, and is ecofriendly (decrease in environmental waste from reduction in plastics and chemical reagents). This chemistry-based multiplexing technology can be easily integrated into a point of care system to expand the capacity and the outreach to resource limited settings. The proposed studies involve development and optimization of Zip- MeltTM DLBCL Classifier Assay, followed by performance characteristics studies in comparison to a Comparator Assay. This Phase I application will establish a platform for detection of gene expression signature to meet the clinical requirement of multianalyte detection in a single workflow.