The study compares the performance of droplet digital PCR (ddPCR) and real-time PCR in quantifying microRNAs (miRNAs). DDPCR demonstrated greater precision (coefficients of variation decreased by 37–86%) and improved day-to-day reproducibility (by a factor of seven) compared to real-time PCR, while maintaining comparable sensitivity. When applied to serum miRNA biomarker analysis, ddPCR showed superior diagnostic performance in identifying individuals with cancer. The study used a hierarchical experimental design with nested replicates and analyzed synthetic miRNAs in water and plasma RNA to assess variation at different stages of the procedure. In clinical serum samples, ddPCR reduced measurement variation by an average factor of seven compared to standard real-time PCR, better resolved cases from controls, and showed higher accuracy in classifying case versus control specimens. The results suggest that ddPCR may be more resilient to differences in sample quality and is more tolerant of PCR inhibitors.The study compares the performance of droplet digital PCR (ddPCR) and real-time PCR in quantifying microRNAs (miRNAs). DDPCR demonstrated greater precision (coefficients of variation decreased by 37–86%) and improved day-to-day reproducibility (by a factor of seven) compared to real-time PCR, while maintaining comparable sensitivity. When applied to serum miRNA biomarker analysis, ddPCR showed superior diagnostic performance in identifying individuals with cancer. The study used a hierarchical experimental design with nested replicates and analyzed synthetic miRNAs in water and plasma RNA to assess variation at different stages of the procedure. In clinical serum samples, ddPCR reduced measurement variation by an average factor of seven compared to standard real-time PCR, better resolved cases from controls, and showed higher accuracy in classifying case versus control specimens. The results suggest that ddPCR may be more resilient to differences in sample quality and is more tolerant of PCR inhibitors.