Contamination tolerances in semiconductor manufacturing processes have changed dramatically in the past two decades, reaching below 20 nm according to the guidelines of the International Technology Roadmap for Semiconductors. The move to narrower line widths drives the need for innovative filtration technologies that can achieve higher particle/contaminant removal performance resulting in cleaner process fluids. Nanoporous filter membrane metrology tools that have been the workhorse over the past decade are also now reaching limits. For example, nanoparticle (NP) challenge testing is commonly applied for assessing particle retention performance of filter membranes. Factors such as high NP size dispersity, low NP detection sensitivity, and high NP particle-filter affinity impose challenges in characterizing the next generation of nanoporous filter membranes. We report a novel bio-surrogate, 5 nm DNA-dendrimer conjugate for evaluating particle retention performance of nanoporous filter membranes. A technique capable of single molecule detection is employed to detect sparse concentration of conjugate in filter permeate, providing >1000- fold higher detection sensitivity than any existing 5 nm-sized particle enumeration technique. This bio-surrogate also offers narrow size distribution, high stability and chemical tunability. This bio-surrogate can discriminate various sub-15 nm pore-rated nanoporous filter membranes based on their particle retention performance. Due to high bio-surrogate detection sensitivity, a lower challenge concentration of bio-surrogate (as compared to other NPs of this size) can be used for filter testing, providing a better representation of customer applications. This new method should provide better understanding of the next generation filter membranes for removing defect-causing contaminants from lithography processes.