The application of chromatography in detecting toxicant-induced systemic alterations remains underexplored. Third-hand smoke (THS) exposure is increasingly recognised as a significant environmental health concern; however, its metabolic and haematological consequences are not yet fully characterised. This study investigates the potential of chromatographic profiling and chemometric analysis as tools for the early detection of metabolic disturbances induced by THS. Fifteen male Wistar rats were allocated to 3 groups: Control and THS exposure (12 or 24 cigarettes per day). Blood samples were collected at multiple time points for haematological analysis, while serum chromatography was conducted on day 10, and lung histology was assessed on day 30. Early metabolic disturbances were observed, characterised by the depletion of key amino acids (L-methionine, DL-lysine, DL-threonine, L-leucine, DL-arginine, and DL-carnitine) and elevated lipid biomarkers (palmitoyl sphingomyelin and phosphatidylcholine) in THS-exposed groups. These metabolic alterations preceded detectable haematological abnormalities. By day 30, haematological impairments and substantial pulmonary damage were evident. The depletion of essential amino acids and elevations in lipid biomarkers may serve as early indicators of toxicant-induced systemic effects. Chromatographic profiling detected metabolic perturbations prior to haematological and pulmonary impairments, highlighting its potential as an early toxicity detection tool. The findings of this exploratory study underscore the promise of chromatography for early toxicity detection, warranting further validation in larger cohorts. © 2025, Walailak University. All rights reserved.