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Hepatitis C is a leading cause of liver disease and transplantation and is a significant burden on public health worldwide. This study aimed to apply the Electronic Nose (E‐Nose) and quadrupole Mass Spectrometry (MS/MS) technologies for screening blood samples from hepatitis C patients and healthy controls. We analysed volatile organic compounds (VOCs) in the headspace over blood samples to identify those VOCs characteristic for diagnosing hepatitis C patients. The study comprised 150 acute hepatitis C patients with age range: 24–59 years, and mean age ±SD: 41.5 ± 12.8 years and 150 age‐matched healthy controls (age range: 24–51 and mean age: 40.11 ± 4.89 years) from the Hospital of the Medical Research Institute, Alexandria University, Alexandria, Egypt. Collected blood samples were analysed qualitatively and quantitatively using the E‐Nose and MS/MS techniques, respectively. Principal component analysis of the E‐Nose 10‐sensor responses accurately classified blood samples from hepatitis C patients and healthy controls. The first two principal components explained over 98.35% of the variance in signals with no false‐positive (healthy controls) or false‐negative (hepatitis C patients) results. MS/MS showed two fragmentation ions at m/z of 104 and 151 Da with the positive electrospray ionization mode (ESI+) in blood samples for hepatitis C patients, but not for healthy controls or background water samples. We identified the two specific fragmentation ions at m/z 104 and m/z 151 Da as malonic acid (MF: C3H4O4; MW: 104.06 g/mol) and monosaccharide pentose (MF: C5H10O5; MW: 150.13 g/mol) in VOCs of the headspace over blood samples for hepatitis C patients. This provides a rationale for developing diagnostic tests for hepatitis C virus based on altered trace VOCs concentrations using the relatively inexpensive, easy‐to‐use, portable and non‐invasive E‐Nose technology. [ABSTRACT FROM AUTHOR]