Immunoglobulin G (IgG) is a specific class of immunoglobulin (Ig) that comprises 75-80% of all Igs in the body. IgGs play a crucial role in adaptive immunity and can provide insights into a patient’s immune response. 1 in 25000 people are affected by dangerously low levels of immunoglobulins, with 39 million people living with Human immunodeficiency virus (HIV). Infection with HIV can lead to acquired immune deficiency syndrome (AIDS). HIV inhibits antibody-producing pathways, resulting in low levels of Igs. An understanding of how to analyse human IgGs is therefore essential for quality assurance and testing of IgG replacement therapeutics. There is currently no method to analyse these complex mixtures of IgGs due to the difficulty of detecting low-abundant IgGs from multiple human plasma samples. To combat this issue, mass spectrometry-based proteomics offers a high-resolution approach for studying IgG composition and diversity. The objective of this study was to optimise a workflow for isolating the IgG fragment antigen-binding (Fab) regions and to enable analysis of IgGs from human IgG-enriched serum samples, allowing assessment of the Fab region subclone distribution. The Fab regions are critical because they contain the variable region, which distinguishes IgG subclones. Differentiation is vital for the body to defend against many different diseases. If IgGs in plasma can be analysed, this would potentially enable the rapid identification of immune complications. Firstly, a method is required to do this. Therefore, IgGs isolated from human plasma were subjected to various sample preparation workflows, including digestion with IdeS and IgdE proteases, to determine which method best characterised the IgG Fab region and to compare the effectiveness of IgG digestion in compatible buffers. 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 from the mass spectrometer (MS). This is due to the need for a large mass of protein to detect low-abundant clones, which the ZenoTOF 7600 is capable of. Advanced mass spectrometric techniques enabled high-resolution separation and identification of IgG subclones, enabling comparative analysis across different samples. The optimised method demonstrated high sensitivity and reproducibility in isolating and digesting IgG subclones. These findings may have implications for IgG product quality assurance and aid further research in the field.