Recent data suggest that passive smoking has a risk comparable to active smoking. Passive smoking is considered dangerous in children and is suspected as a cause of asthma. However, some reports are opposing such claims, indicating the need for solid results and large-scale studies. This scientific work aims to develop a method for the determination of nicotine (NCOT) and major nicotine’s metabolite cotinine (COT) in urine samples, using gas chromatography-mass spectrometry (GC-MS). For sample preparation, liquid-liquid extraction was applied after an optimization study with different extraction media. Eventually, 1 mL of dichloromethane was selected for the extraction of 0.5 mL of urine. Suitable chromatographic conditions were found for the rapid and accurate determination of NCOT and COT. The method was validated to meet the criteria for application in a bioanalytical laboratory. Injection of 2 μL was done in GC/MS, and SIM (selected ion monitoring) analysis was performed with the following ions (m/z): 162 (quantifier ion) and 84, 133, 161 qualifier ions for NCOT, and 176 (quantifier ion) and 98, 118, 119, 147 qualifier ions for COT. Nicotine-D4 (NCOT-D4) and cotinine-D3 (COT-D3) and were used as internal standards with quantifier ions 101 and 166, respectively. The retention time (Rt) for NCOT was 7.557 min and for COT 9.743 min. The duration of the GC-MS analysis was 20 minutes. The method showed a linear dynamic range from 0.5 to 50 ng/mL. Finally, the method was applied to the analysis of sixty clinical pediatric samples obtained from Aristotle University’s pediatric clinic to check for possible exposure to smoke.