Exogenous progesterone has several applications in human health and in veterinary medicine, especially in fixed-time artificial insemination protocol. Progesterone nanoencapsulation in biocompatible polymers, such as poly(methyl methacrylate) (PMMA), is an alternative to substitute silicone-based release device traditionally used for estrus control. Progesterone concentration inside the nanoparticles must be precisely known; for that reason, a validation methodology must be applied to ensure reliable results, suitable for nanoparticles application. In this work, an UV-Vis spectrophotometric method was validated for the determination of progesterone in PMMA nanoparticles synthesized by miniemulsion polymerization. Chloroform was used as solvent, showing selectivity to the encapsulated drug and the components of the polymeric matrix did not influence progesterone recovery. Detection and quantitation limits (DL and QL) obtained were 0.32 and 0.96 mg·L−1, respectively, and precision tests (between different analysts and equipment) indicated acceptable Relative Standard Deviations (RSD < 5%). Miniemulsion polymerization reactions were carried out producing two different morphologies: nanospheres (NS) and nanocapsules (NC), with average intensity diameters (Dz) of 150–200 nm and 240–300 nm, respectively. Polymerization gravimetric conversions obtained for both cases were higher than 95% and encapsulation efficiencies greater than 69% and 90% for the nanospheres and nanocapsules, respectively