Monitoring nicotine and its metabolites in bodily fluids is crucial for assessing tobacco exposure and related toxicity in clinical practice. Due to its high sensitivity for detecting low-level analytes, liquid chromatography–tandem mass spectrometry (LC–MS/MS) was employed to enhance a robust and accurate analytical procedure for quantifying nicotine and its derivatives in serum, urine, and saliva. Comparative analysis was conducted on samples collected from individuals with and without smoking habits. The LC–MS/MS method utilized an API 3200 triple quadrupole instrument paired with a Phenomenex Luna C18 column. Method sensitivity was demonstrated through evaluation of sensitivity parameters, including LOD and LLOQ for each analyte in all matrices. Inter-assay variability and bias values at the LLOQ level were kept below 20%. In serum, the LOD–LLOQ ranges were 0.02–0.05 μg/L for nicotine, 0.32–0.95 μg/L for cotinine, 0.07–0.25 μg/L for 3-OH cotinine, and 0.22–0.69 μg/L for norcotinine. In urine, the respective values were 0.03–0.10, 0.23–0.82, 0.06–0.14, and 0.20–0.55 μg/L, and in saliva, the values were 0.12–1.59, 0.22–2.34, 0.10–0.17, and 0.33–3.01 μg/L. The proposed method is efficient, specific, and adaptable to routine clinical workflows, providing a valuable tool for monitoring tobacco-related exposure.