Brazil is the largest producer and exporter of orange in the world, with 50% of world´s production and 85% of the world´s exports. Fungi are responsible for large post-harvest losses in various commodities. In particular the fungus Penicillium digitatum, which is responsible for up to 90% of the total losses to the brazilian citrus in post-harvest. During infection by this phytopathogen to the citrus host, the alkaloids of tryptoquialanines type A and B, are synthesized in vivo, which could present a problem for the world's population, once mycotoxins produced by fungi in general are notoriously toxic. These compounds have still unknown effects in literature. The aim of this study was to develop and validate an analytical method for determination of tryptoquialanines A and B synthesized by P.digitatum in different layers of orange fruits (epicarp, mesocarp and endocarp), aiming to understand the diffusion of these mycotoxins between citrus layers, as well as its derivatives from fruit. In this sense, both alkaloid standards were produced by P.digitatum in vitro and purified. Purification was performed by HPLC-preparative and the compounds were characterized by NMR techniques (1H, HMQC, HMBC). Both alkaloid standards were purified and then used to develop and validate the method which was employed in the extraction and clean up QuEChERS with some modifications, and LC-ESI-MSMS was used to quantify. A good linearity was obtained under a range of 0.025-0.25 mg/kg with r2 >0.99 except for the flavedo is which presented r2 >0.98 to tryptoquialanine B due to the matrix effect. The limits of detection (LOD) and quantification (LOQ) were 4.7 µg/kg and 15.7 µg/kg respectively. The relative standard deviations (RSDs) were ≤ 18% during the validation process. The developed analytical method can be used for the routine monitoring of the P.digitatum mycotoxins in citrus.