The study compares droplet digital PCR (ddPCR) and real-time PCR for absolute quantification of microRNAs (miRNAs). ddPCR offers higher precision and day-to-day reproducibility, with a 37–86% reduction in coefficient of variation (CV) compared to real-time PCR. It also provides more accurate quantification of miRNAs in serum samples, leading to improved diagnostic performance for cancer detection. ddPCR is less sensitive than real-time PCR for some miRNAs but is more robust to variations in PCR efficiency and sample quality. The study used synthetic miRNAs and clinical serum samples to evaluate performance, finding that ddPCR consistently showed lower variation across all miRNAs tested. ddPCR also demonstrated better resolution of cases from controls in clinical samples, with statistically significant differences in miR-141 abundance. The study highlights the advantages of ddPCR for high-precision nucleic acid quantification, particularly in complex biological matrices. The results suggest that ddPCR is a promising tool for absolute quantification of miRNAs and other nucleic acids in clinical and research settings.The study compares droplet digital PCR (ddPCR) and real-time PCR for absolute quantification of microRNAs (miRNAs). ddPCR offers higher precision and day-to-day reproducibility, with a 37–86% reduction in coefficient of variation (CV) compared to real-time PCR. It also provides more accurate quantification of miRNAs in serum samples, leading to improved diagnostic performance for cancer detection. ddPCR is less sensitive than real-time PCR for some miRNAs but is more robust to variations in PCR efficiency and sample quality. The study used synthetic miRNAs and clinical serum samples to evaluate performance, finding that ddPCR consistently showed lower variation across all miRNAs tested. ddPCR also demonstrated better resolution of cases from controls in clinical samples, with statistically significant differences in miR-141 abundance. The study highlights the advantages of ddPCR for high-precision nucleic acid quantification, particularly in complex biological matrices. The results suggest that ddPCR is a promising tool for absolute quantification of miRNAs and other nucleic acids in clinical and research settings.