Background: Thymus vulgaris essential oil (Tv-EO) is known to have antibacterial, antifungal, and antioxidant activities. Encapsulation of Tv-EO in polymeric nanoparticles (NPs) can prevent volatilization of its components and can provide protection against external agents. Under these circumstances, it is crucial to assure the presence and quantity of the Tv-EO components (γ-terpinene, thymol, and carvacrol) in the NPs. Objective: To determine the chemical composition and physicochemical characterization of Tv-EO as well as develop and validate a HSPM-gas chromatography (GC) method for the analysis of Tv-EO components encapsulated in NPs. Materials and Methods: Tv-EO was characterized by physicochemical analysis for relative density, refractive index, and optical rotation and analyzed by GC flame ionization detector and GC-mass spectrometry. The headspace–solid-phase microextraction-gas chromatography (HS-SPME-CG) validation was assessed, Tv-EO-NPs were prepared by nanoprecipitation, and its properties were determined by photon correlation spectroscopy. Results: Tv-EO was characterized by physicochemical analysis for relative density (0.934 g/cm3), refraction index (1.559), and optical rotation (−0.084°). Seventeen components were identified in Tv-EO; among these, the sesquiterpenes, thymol (34.28%), o-cymene (31.78%) and γ-terpinene (13.22%). The method was validated for linearity (R2 ≥ 0.99), precision (intraday 7.02, 10.33, and 8.60 and inter-day 10.60, 10.60, and 10.99), accuracy (99.35, 109.4, and 98.84%) and robustness for γ-terpinene, thymol and carvacrol, respectively. The limit of detection and limit of quantification were calculated as 0.69, 0.40, and 0.39 μg/mL and 2.11, 1.22, and 1.20 μg/mL for γ-terpinene, thymol, and carvacrol, respectively. An encapsulation percentage of 47.51% of total essential oil was obtained. Conclusion: The experimental data show that HS-SPME reduces interference of the NP-matrix and concentrates the Tv-EO components. HS-SPME-CG can be considered as a good alternative to the already existing methods for analysis of essential oil encapsulated in NPs.