Twenty-four samples of heroin from different illicit drug seizures were analyzed using proton Nuclear Magnetic Resonance (1H NMR) and two-dimensional diffusion-ordered spectroscopy (2D DOSY) 1H NMR. A careful assignment and quantification of 1H signals enabled a comprehensive characterization of the substances present in the samples investigated: heroin, its main related impurities (6-acetylmorphine, acetylcodeine, morphine, noscapine and papaverine) and cutting agents (caffeine and acetaminophen in nearly all samples as well as lactose, lidocaine, mannitol, piracetam in one sample only), and hence to establish their spectral signatures. The good agreement between the amounts of heroin, noscapine, caffeine and acetaminophen determined by 1H NMR and gas chromatography, the reference method in forensic laboratories, demonstrates the validity of the 1H NMR technique. In this paper, 2D DOSY 1H NMR offers a new approach for a whole characterization of the various components of these complex mixtures.