Immunoglobulin G (IgG) plays a crucial role in adaptive immunity and can provide insights into patient’s immune response. Mass spectrometry-based proteomics offers a high-resolution approach to studying IgG composition and diversity. The objective of this study was to optimise a middle-down proteomic workflow for isolating and ability to analyse IgGs from human serum to allow for the assessment of IgG Fab region subclone distribution. Efficient analysis of IgG in serum will enable the rapid identification of immune complications. IgGs were isolated from human serum using magnetic affinity beads and subjected to various sample preparation workflows, including full and partial reduction, digestion with IdeS and IgdE to determine which method allowed for the best characterisation of the IgG Fab region. Due to the complexity of the samples, the ZenoTOF 7600 mass spectrometer was used to validate the method's effectiveness in generating high-quality data.  Advanced mass spectrometric techniques enabled high-resolution separation and identification of IgG subtypes, which will allow a comparative analysis between different samples. The optimised method demonstrated high sensitivity and reproducibility in isolating and digesting IgG subclones. These findings may have implications for understanding population-specific immune responses and could inform the development of targeted immunotherapies and vaccine strategies.