<p>Quantification of cellular protein levels is a central aspect of molecular biology. Although large-scale proteomic quantification technologies have been developed, Western blotting remains a widely used method for researchers investigating a limited number of target proteins. As the demand for rigorous statistical evaluation of Western blotting data increases, particularly in studies involving larger sample sizes, the limitations imposed by the finite number of samples that can be loaded onto a single SDS-PAGE gel have become more apparent. Consequently, the quantitative capacity of Western blotting experiments is often restricted. To address this, we developed an enhanced method for quantitative Western blotting using a multi-gel blotting approach, which was originally described by Aksamitiene et al. for high-throughput Western blotting, wherein multiple SDS-PAGE gels are simultaneously transferred onto a single PVDF membrane. To optimize the accuracy of quantification, transfer conditions were systematically examined, and a standard protocol with which the majority of protein is uniformly transferred to a membrane is provided. Furthermore, considering the inherent non-linear relationship of protein concentration and Western blotting signal intensity, we adopted a log-logistic regression model to calibrate the relationship for the estimation of protein concentrations in unknown samples. Taken together, an enhanced quantitative Western blotting method is provided, offering improved accuracy and reliability in protein quantification.</p>