ABSTRACT A specific HS-GC-MS method has been developed, optimized and validated for the determination of five genotoxic impurities namely Nitromethane, Nitroethane, 2-Nitropropane, 1-Nitropropane and 1-Nitrobutane in TRB-G, intermediate of Trabectedin drug substance. Chromatographic separation of five genotoxic impurities was achieved on Capillary GC column (Rtx-1701. Fused silica capillary column; 30 m length; 0.25mm internal diameter. coated with 14% Cyanopropylphenyl and 86% dimethyl polysiloxane stationary phase of 1.0 µm film thickness) and passing helium carrier gas with Electron Impact ionization (EI) in Selective Ion Monitoring (SIM). The mass fragments (m/z) were selected for the quantification of m/z-30 for Nitromethane, m/z-29 for Nitroethane, m/z-43 for 2-Nitropropane and 1-Nitropropane and m/z-57 for 1-Nitrobutane. The performance of the method was assessed by evaluating the specificity, linearity, sensitivity, precision and accuracy experiments. The established limit of detection values for the genotoxic impurities were in the range of 0.25 µg/g – 0.58 µg/g and limit of quantification values were in the range of 0.75 µg/g – 1.75 µg/g. This developed method was found to be linear with correlation coefficient is greater than 0.999. The average recoveries for the accuracy were in the range of 89.4–106.8%. Developmental and validation experiments were discussed in detail in this paper. Keywords: Trabectedin (TRB), HS-GC-MS, Genotoxic impurities and Method validation.