This article presents a standard curve-based method for processing relative real-time PCR data, which is a reliable and simple alternative to PCR efficiency-based calculations. The method avoids the complexities and potential issues associated with PCR efficiency assessment, such as the variability in efficiency over the PCR run and the need for multiple dilutions. The procedure includes steps for noise filtering, threshold selection, crossing point calculation, statistical assessment of intra-assay variation, and derivation of non-normalized values from crossing points. The authors validate the method using breast cancer biopsies and demonstrate its effectiveness through computer simulations. The standard curve approach is found to be robust and suitable for routine laboratory practice, especially when low dispersion in PCR replicates is observed. The article also discusses the limitations of amplitude normalization and provides recommendations for its use. Overall, the standard curve method offers a straightforward and accurate approach to relative real-time PCR data processing.This article presents a standard curve-based method for processing relative real-time PCR data, which is a reliable and simple alternative to PCR efficiency-based calculations. The method avoids the complexities and potential issues associated with PCR efficiency assessment, such as the variability in efficiency over the PCR run and the need for multiple dilutions. The procedure includes steps for noise filtering, threshold selection, crossing point calculation, statistical assessment of intra-assay variation, and derivation of non-normalized values from crossing points. The authors validate the method using breast cancer biopsies and demonstrate its effectiveness through computer simulations. The standard curve approach is found to be robust and suitable for routine laboratory practice, especially when low dispersion in PCR replicates is observed. The article also discusses the limitations of amplitude normalization and provides recommendations for its use. Overall, the standard curve method offers a straightforward and accurate approach to relative real-time PCR data processing.