Lipid nanoparticles serve as drug delivery vehicles for biopharmaceutical products. The lipid membrane shields internal nucleic-acid drug cargo from enzymatic degradation and facilitates cellular uptake of the drug. However, existing methods to assess drug loading within liposomes are limited to averaged bulk measurements, which obscures heterogeneity of the biopharmaceutical formulation. This report describes the development of a single-liposome analysis method to measure copy numbers of DNA within liposomes and assess population heterogeneity. This novel measurement was achieved by integrating two orthogonal polymerase chain reaction (PCR) techniques─digital PCR (dPCR) and quantitative PCR (qPCR)─within a single microfluidic assay. The dPCR dimension quantified liposomes to validate their capture in the single-liposome analysis regime. The qPCR dimension quantified DNA copy numbers packaged within each liposome. The ability of digital quantitative PCR (dqPCR) to analyze large numbers of individual liposomes in parallel revealed significant population heterogeneity, which could not be obtained from standard bulk analysis methods. Our innovative measurement of internal DNA cargo from single liposomes has the potential to inform liposome synthesis procedures and create more uniform liposomal biopharmaceutical formulations to enhance drug safety and efficacy.