The formation of a biomolecular corona significantly influences the biological identity and behavior of nanomaterials, affecting cellular uptake, toxicity, and biodistribution. Protein analysis is a central tool in corona characterization, providing insight into the composition and affinity of proteins adsorbed onto nanomaterial surfaces. However, standard protocols often fail to account for the unique physicochemical properties of advanced nanomaterials, such as graphene oxide (GO), which can interfere with protein elution and lead to biased or incomplete analyses. To address these challenges, we investigated the limitations of conventional SDS-based elution methods for protein corona analysis on GO. We compared these to an improved protocol utilizing chaotropic agents, urea, and thiourea in a stepwise extraction process. Protein corona complexes were isolated after incubation with GO and subjected to sequential protein elution using SDS-based or urea/thiourea-based buffers prior to SDS-PAGE and LC-MS/MS analysis. Our findings revealed that SDS-based protocols were insufficient for desorbing strongly bound proteins, particularly those with hydrophobic characteristics. The use of chaotropic agents significantly improved protein recovery, enabling near-complete elution from the GO surface. Importantly, the improved method revealed protein populations underrepresented in standard protocols, demonstrating its enhanced capability to recover high-affinity, hydrophobic corona constituents. This study underscores the need to tailor biomolecular corona characterization protocols to specific material properties. The improved method offers a more accurate and reproducible strategy for analyzing the protein corona on GO, addressing critical gaps in current nanosafety practices. Standardizing such optimized protocols is essential for ensuring the reliability of in vitro assessments and for promoting the safe and sustainable application of Ad-NMs in biological and environmental contexts.