Objectives: The objective of this study was to develop a method for simultaneous concentration determination of nicotine and its metabolisms in biological urine sample by making calibration curves using reference standard solutions of known concentrations to calculate unknown concentrations in urine samples. Determine the validation parameters of the specified method. Design: Experimental study Settings: Laboratory of Toxicology, Department of pathology, forensic medicine and cytology, University Hospital of Split. Material and Methods: Calibration solutions were obtained by serial dilution of the reference standards, first in methanol and then in a urine sample, after which equal volumes of the same concentrations of all three substances were taken and a larger concentration area was covered. MIX solutions of the standards were extracted by liquid-liquid (LLE) method, diluted with chloroform and analyzed by total ion current (TIC) gas chromatography with mass spectrometer. After the preparation of the calibration curves, their application is followed in the determination of the concentration of nicotine and metabolites in real urine samples. Results: Calibration curves for nicotine, cotinine, and 3-OH-cotinine were successfully constructed using the obtained signals by the targeted measurement of standard solutions in urine at concentrations of 0.3-1000 ng/mL. The linearity of the method with a correlation coefficient R> 0.99 was proved. Repeatability also meets the eligibility criterion (RSD <10%). The mass concentrations of the detected nicotine and its metabolites in samples of unknown concentrations were calculated using previously prepared calibration curves. Conclusion: The results of this study confirmed the choice of GC-MS technique in combination with previous LLE extraction of desired analytes for the quantitative determination of nicotine and its metabolites in biological urine samples. The validation factors performed satisfy the eligibility criterion. The presence of nicotine and metabolites in non-smokers has also been demonstrated, demonstrating the high impact of secondhand smoke on humans. Further studies should include a large number of nonsmokers and various conditions of exposure to secondhand smoke.