Presented here is the optimization and development of a desorption electrospray ionization mass spectrometry (DESI-MS) method for detecting natural products on tissue surfaces. Bromophycolides are algal diterpene-benzoate macrolide natural products that have been shown to inhibit growth of the marine fungal pathogen Lindra thalassiae. As such, they have been implicated in antimicrobial chemical defense. However, the defense mechanisms are not yet completely understood. Precise detection of these compounds on algal tissue surfaces under ambient conditions without any disruptive sample processing could shed more light onto the processes involved in chemical defense of marine organisms. Conventional DESI-MS directly on algal tissue showed relatively low sensitivity for bromophycolide detection. Sensitivity was greatly improved by the addition of various anions including Cl(-), Br(-), and CF(3)COO(-) into the DESI spray solvent. Chloride adduction gave the highest sensitivity for all assayed anions. Density functional optimization of the bromophycolide anionic complexes produced during DESI supported this observation by showing that the chloride complex has the most favorable binding energy. Optimized DESI protocols allowed the direct and unambiguous detection of bromophycolides, including A, B, and E, from the surface of untreated algal tissue.