High mobility group box 1 (HMGB1) is a chromatin-associated protein that is also secreted from activated immune cells, necrotic cells, and certain cancer cells. Previous studies have described that different patterns of post-translational modification (PTM), particularly acetylation and oxidation, mediate HMGB1 release and confer distinct extracellular HMGB1 signaling activity. However, the regulation of HMGB1 subcellular mobility in lung cancer by PTMs has not yet been established. To this end, a methodology was developed to quantify HMGB1 lysine acetylation and cysteine oxidation in several lung cancer models by a two-dimensional nano-ultrahigh-performance liquid chromatography parallel reaction monitoring/high-resolution mass spectrometry (2D-nano-UHPLC-PRM/HRMS) platform. Secreted HMGB1 was immunoprecipitated with a stable isotope-labeled HMGB1 internal standard followed by CD3-acetylation and protease digestion in order to quantify site-specific acetylation on secreted HMGB1. A similar approach including differential alkylation of HMGB1’s cysteine residues was used to quantify site-specific cysteine oxidation on secreted HMGB1. Cisplatin, but not carboplatin, induced secretion of HMGB1 from human non-small cell lung cancer (NSCLC) A549 cells in a necrosis-independent mechanism. Cisplatin-mediated HMGB1 secretion was dose-dependent and was regulated by nuclear exportin 1 (XPO1) also known as chromosomal maintenance 1 (CRM1) rather than acetylation or oxidation. Malignant mesothelioma (MM) cell lines were found to constitutively secrete unacetylated HMGB1, and the cysteine oxidation profiles in HMGB1 from cisplatin-mediated, dimethyl sulfoxide-mediated, and MM secretion were found to have statistically significant differences. These findings suggest that inhibition of XPO1 could potentiate the anti-tumor activity of cisplatin by increasing nuclear accumulation of HMGB1, an inhibitor of cisplatin DNA-adduct repair. Furthermore, low-dose cisplatin therapy could modulate the immune response in NSCLC through the established chemokine activity of extracellular reduced HMGB1. Differences between NSCLC and MM in their relationship with immune system stimulation could explain in part why they differ in basal HMGB1 secretion despite releasing similar HMGB1 proteoforms.