DIA (Data Independent Acquisition) analysis is utilized to accurately identify and quantitatively measure proteins and other biological molecules in large-scale biological samples. It provides a more comprehensive and in-depth analysis, offering rapid and precise information in complex biological systems. Moreover, the FAIMS (high-field asymmetric waveform ion mobility spectrometer) interface reduces chemical noise and improves signal-to-noise ratios, particularly benefiting proteomics. To enhance the utility of DIA in the diagnostic field, we aimed to couple it with a FAIMS interface to maximize protein identification in human serum and optimize quantitative comparative analysis. Initially, we validated the effectiveness of DIA analysis before applying it to human serum and conducted CV (compensation voltage) optimization. Subsequently, we confirmed whether these results were consistent in human serum and compared the quantitative results of DIA-FAIMS with or without depletion of high abundant proteins. By integrating multiple results, we established an optimized method for human serum analysis that maximizes effectiveness.