Desorption electrospray ionization (DESI) mass spectrometry is applied to the in situ detection of alkaloids in the tissue of poison hemlock (Conium maculatum), jimsonweed (Datura stramonium) and deadly nightshade (Atropa belladonna). The experiment is carried out by electrospraying micro-droplets of solvent onto native or freshly-cut plant tissue surfaces. No sample preparation is required and the mass spectra are recorded under ambient conditions, in times of a few seconds. The impact of the sprayed droplets on the surface produces gaseous ions from organic compounds originally present in the plant tissue. The effects of operating parameters, including the electrospray high voltage, heated capillary temperature, the solvent infusion rate and the carrier gas pressure on analytical performance are evaluated and optimized. Different types of plant material are analyzed including seeds, stems, leaves, roots and flowers. All the previously reported alkaloids have been detected in C. maculatum, while fifteen out of nineteen known alkaloids for D. stramonium and the principal alkaloids of A. belladonna were also identified. All identifications were confirmed by tandem mass spectrometry. Results obtained show similar mass spectra, number of alkaloids, and signal intensities to those obtained when extraction and separation processes are performed prior to mass spectrometric analysis. Evidence is provided that DESI ionization occurs by both a gas-phase ionization process and by a droplet pick-up mechanism. Quantitative precision of DESI is compared with conventional electrospray ionization mass spectrometry (after sample workup) and the RSD values for the same set of 25 dicotyledonous C. maculatum seeds (one half of each seed analyzed by ESI and the other by DESI) are 9.8% and 5.2%, respectively.