Analyzing qPCR data is not as straight-forward as it seems. Many online qPCR analysis tools exist, but the majority does not take into account the variables that determine the quality of qPCR measurements or use the unreliable ΔΔCt method. Together with the AMC department of Clinical Epidemiology we developed a sophisticated qPCR analysis pipeline. Our approach includes state-of-the-art quality control algorithms and interplate corrections, combined with a PCR-efficiency dependent calculation of the starting amount of the biomarker. Our analysis method guarantees reliable and reproducible results.