A novel, simple and precise headspace gas chromatography-mass spectrometry (HS-GC-MS) method was developed and validated for the determination of acetyl chloride (AC) in various anti-cancer drug substances like bendamustine HCl, Ibrutinib, trifluridine and abemaciclib drug substances. In this methodology, AC reacts with isopropyl alcohol and is converted into isopropyl acetate. Hence AC is quantified as Isopropyl acetate. The lower level of detection was achieved on the capillary GC column (DB-624, Fused silica capillary column; 30 m length; 0.32 mm internal diameter, coated with 6% Cyanopropylphenyl and 94% dimethyl polysiloxane stationary phase of 1.8 μm film thickness) with electron impact ionization (EI) technique by GC-MS in selective ion monitoring (SIM) mode. The mass ions were selected for the quantifier is 63 and the qualifier is 87 for isopropyl acetate. The performance of the method was assessed by evaluating the specificity, linearity, sensitivity, precision, accuracy and robustness experiments. The established limit of detection and limit of quantification values for the AC were 0.1 and 0.3 μg/g, respectively. This developed method was found to be linear with a correlation coefficient is greater than 0.999 for all four drug substances. The average recoveries of AC in bendamustine HCl, ibrutinib, trifluridine and abemaciclib were obtained 106.1, 109.0, 97.4 and 101.6%, respectively. The proposed method was validated successfully as per ICH guidelines. Hence, the proposed method can be routinely used for the quantification of AC in various anti-cancer drug substances.