This work represents the first reporting of a comprehensive bioanalytical GLP methodology detailing the mass spectrometric quantitation of PF-05212384 dosed as a targeted polymeric encapsulated nanoparticle (PF-07034663) to monkeys. Polymeric nanoparticles are a type of drug formulation that enables the sustained release of an active therapeutic agent (payload) for targeted delivery to specific sites of action such as cancer cells. Through the careful design and engineering of the nanoparticle formulation, it is possible to improve the biodistribution and safety of a given therapeutic payload in circulation. However, the bioanalysis of nanoparticles is challenging due to the complexity of the nanoparticle drug formulation itself and the number of pharmacokinetic end points needed to characterize the in vivo exposure of the nanoparticles. Gedatolisib, also known as PF-05212384, was reformulated as an encapsulated targeted polymeric nanoparticle. The bioanalytical assays were validated to quantitate both total and released PF-05212384 derived from the encapsulated nanoparticle (PF-07034663). Assay performance calculated from quality control samples in three batch runs demonstrated intraday precision and accuracy within 10.3 and 12.2%, respectively, and interday precision and accuracy within 9.1 and 8.5%, respectively. This method leveraged automation to ease the burden of a laborious and complicated sample pretreatment and extraction procedure. The automated method was used to support a preclinical safety study in monkeys in which both released and total PF-05212384 concentrations were determined in over 1600 monkey plasma study samples via LC-MS/MS.