The article evaluates the performance of two label-free methods, peak area intensity measurements and spectral counting, for quantifying protein abundance changes in shotgun proteomic analyses. The authors developed Serac software to assess the effectiveness of these methods. Peak area intensity measurements showed good linearity and agreement with expected protein ratios after normalization and background subtraction. Spectral counting, which compares the number of MS/MS spectra assigned to each protein, also demonstrated linearity and high correlation with protein concentrations. The study tested these methods using standard proteins spiked into complex samples and found that peak area intensity measurements were more sensitive, while spectral counting provided more accurate estimates of protein ratios. The methods were applied to analyze differential protein expression in human erythroleukemia K562 cells stimulated with mitogen-activated protein kinase pathway activators, showing good correlations between protein and mRNA expression changes. Overall, the study highlights the complementary nature of these methods in profiling protein abundance changes in biological systems.The article evaluates the performance of two label-free methods, peak area intensity measurements and spectral counting, for quantifying protein abundance changes in shotgun proteomic analyses. The authors developed Serac software to assess the effectiveness of these methods. Peak area intensity measurements showed good linearity and agreement with expected protein ratios after normalization and background subtraction. Spectral counting, which compares the number of MS/MS spectra assigned to each protein, also demonstrated linearity and high correlation with protein concentrations. The study tested these methods using standard proteins spiked into complex samples and found that peak area intensity measurements were more sensitive, while spectral counting provided more accurate estimates of protein ratios. The methods were applied to analyze differential protein expression in human erythroleukemia K562 cells stimulated with mitogen-activated protein kinase pathway activators, showing good correlations between protein and mRNA expression changes. Overall, the study highlights the complementary nature of these methods in profiling protein abundance changes in biological systems.