It is well established that the biomolecular corona affects the biological behavior of nanomaterials, including cellular uptake, toxicity, and biodistribution. However, the unique physicochemical properties of advanced materials, such as graphene oxide, challenge the effectiveness of standard protocols for biomolecular corona characterization, which may lead to incomplete biomolecule recovery and biased experimental results. Protein analysis is one of the broadest techniques in the characterization of the biomolecular corona, providing important information about the composition and behavior of proteins adsorbed onto nanomaterial surfaces. Two widely accepted protein analysis methods include SDS-PAGE and mass spectrometry, and both require the complete elution of the proteins from the nanoparticle surface during denaturation steps. In this work, limitations of widely used SDS-based elution methods with GO were identified, and an improved protocol using chaotropic agents (urea and thiourea) was developed. The stepwise extraction allowed for near-complete protein desorption. Under the modified protocol, strongly bound proteins that are more hydrophobic have been proved to be underestimated using the standard method. This further reiterates the necessity for the development of methodologies tailored to the specific materials under study which accurately characterize the protein corona. Our results highlight the need for standardization and optimization of protocols to ensure reproducibility and reliability in nanosafety studies, hence promoting the safe and sustainable use of advanced materials in biological and environmental systems.