This article addresses the importance of baseline estimation in the analysis of quantitative PCR (qPCR) data, highlighting that baseline estimation errors directly affect PCR efficiency values and propagate exponentially into the estimated starting concentrations and fold-difference results. The authors developed an algorithm to estimate the baseline by reconstructing the log-linear phase from the early plateau phase of the PCR reaction, which significantly reduces variability and bias in qPCR results. The algorithm is based on the assumption that PCR efficiency is constant from the first cycle onward, and it is applied to datasets from different qPCR platforms. The study demonstrates that using the mean PCR efficiency per amplicon, rather than individual sample efficiencies, reduces variability and bias in gene expression ratios. The article also discusses the limitations of standard curve-derived PCR efficiency values and nonlinear analysis procedures, emphasizing the importance of proper baseline handling in qPCR data analysis.This article addresses the importance of baseline estimation in the analysis of quantitative PCR (qPCR) data, highlighting that baseline estimation errors directly affect PCR efficiency values and propagate exponentially into the estimated starting concentrations and fold-difference results. The authors developed an algorithm to estimate the baseline by reconstructing the log-linear phase from the early plateau phase of the PCR reaction, which significantly reduces variability and bias in qPCR results. The algorithm is based on the assumption that PCR efficiency is constant from the first cycle onward, and it is applied to datasets from different qPCR platforms. The study demonstrates that using the mean PCR efficiency per amplicon, rather than individual sample efficiencies, reduces variability and bias in gene expression ratios. The article also discusses the limitations of standard curve-derived PCR efficiency values and nonlinear analysis procedures, emphasizing the importance of proper baseline handling in qPCR data analysis.