The quantitation of the major capsid protein L1 is an important metric during the pharmaceutical manufacturing of human papilloma virus (HPV) vaccines, as they are critical components of virus like particles (VLPs) that form the core of the drug product. During the production of VLPs, the L1 protein is present in multiple states, including monomer, multimer, fully formed VLPs and aggregate species, whose expression levels provides an important read-out of upstream productivity and downstream purification efficiency through the measurement of step yields. However, quantitation of total L1 protein is challenging not only due to its presence in multiple states, but also due to the matrix complexity and purification stage of the samples, which spans complex cell lysate to cleaner post purification material. Current analytical methods typically implemented for L1 quantitation includes direct UV measurement (such as SoloVPE), which is robust and easily deployed, but best suited to analysis of purified samples. Automated capillary electrophoresis techniques such as Simple Western are well established but dependent on reproducible binding to accessible L1 epitopes and potentially susceptible to antibody lot to lot reproducibility which may pose an operational risk. Mass spectrometry-based techniques provide excellent sensitivity and characterization advantages but are challenging to deploy in a manufacturing setting. Additionally, conventional one-dimensional liquid chromatography separation of L1 from host cell protein or cellular components is ineffective particularly in high-complexity lysate samples and intermediates prior to chromatographic purification steps. Herein, we present a sample preparation strategy and analytical method that is capable of total L1 quantitation regardless of its multimeric state and is compatible with sample matrices ranging from crude lysate to purified samples, without the use of complicated and serotype-specific reagents. We employ reduction and heat-denaturation during sample preparation to simplify the multimeric states of L1 to its monomer form and utilize two-dimension liquid chromatography (2D-LC) with first dimension (