Annonaceous acetogenins (AAGs) are environmental neurotoxins from the fruit pulp of several Annonaceae species, whose consumption was linked to the occurrence of sporadic atypical Parkinsonism with dementia. The quantification of the prototypical AAG annonacin in Rat brain homogenates was performed by UPLC-MS/MS in selected reaction monitoring (SRM) mode, using a triple quadrupole mass analyzer. A natural analog of annonacin was used as an internal standard. Analyzed data set of the analytical validation of this method is presented, including stability of the samples, extraction recovery and matrix effect, supporting the results described in the article “Quantification of the environmental neurotoxin annonacin in Rat brain by UPLC-MS/MS” .