The main goal of this work was the development of spectrofluorimetric and reversed-phase high-performance liquid chromatography (RP-HPLC) methods for citrus essential oils (CEO) analysis. For this purpose, surfactant-free microemulsions (SFMEs) were used as an approach for sample treatment. First, the SFME formation region was studied at different water:EO weight proportions (1:4, 1:2, 1:1, 2:1, 4:1 w/w) in the presence of propan-1-ol and octan-1-ol (10:3 w/w). Conductometric titrations indicated micellar aggregates containing EO (oil-in-water microemulsion) for the 4:1 proportion (droplets of hydrodynamic radius of 95.7 ± 5.3 nm). In such conditions, fluorescence increased allowing the use of 3D fluorescence spectroscopy to obtain spectroscopic fingerprint pattern that was used aiming discriminant analysis, considering nine EO Brazilian brands, along with unfold principal component analysis(UPCA). A cumulative variance of 96.7 percent was obtained for the first three principal components and score plots showed distinct location for each group. Preliminary study showed the capability of these systems in evaluating storage conditions, and adulteration. The impact of storage conditions was made over 21 days exposed to light with results showing large spectral differences compared to a sample stored in amber flask at 22 degrees C. Adulteration of EOs by canola and mineral oil fortification was detected at different levels (1, 5, 10 and 20 percent, w/w) and, in addition to the spectral differences, a change in microemulsion stability was observed. In a second stage of the study, these systems were replicated for different CEOs, including sweet and sour orange, tangerine, lemon, and grapefruit. Studies were employed to assess optimal conditions for system formation, aiming to encompass all evaluated CEOs without compromising measurement reproducibility. The new SFME condition adopted was 15 microL of the oily phase containing EO and octan-1-ol (1:2 v/v), 19 mL of water, and propan-1-ol up to the 25 mL final volume. In addition to low sample consumption, a significant increase in fluorescence was achieved, despite the lower proportion of the oily phase. Excitation-emission matrix data were utilized for clustering analysis. UPCA was successfully applied, with cumulative variance of 99.5 percent for the first three principal components. Eigenvectors decomposition revealed a significant influence of the 336/436 nm excitation/emission wavelengths. Complementary analyses by HPLC confirm the relationship between fluorophores and the non-volatile fraction, characteristic of CEOs. The combination of low sample consumption and high-water content makes the application of these systems advantageous for CEO differentiation. Transparency and low viscosity are also considered positive aspects. In a third and final stage, the effect of the sampling medium on the analysis of polymethoxyflavones (PMFs) in sweet orange EO by RP-HPLC was evaluated, utilizing the knowledge acquired in previous stages regarding the formation of SFME systems. This study was focused on analyzing PMFs present in the non-volatile fraction of sweet orange EO, which includes tetra-O-methyl-scutellarein, sinensetin, tangeretin, nobiletin, and heptamethoxyflavone. Two methods utilizing isocratic elution with different mobile phases, (A) water/methanol and (B) water/acetonitrile, were employed using absorciometric and fluorimetric detection. The sampling medium significantly influenced chromatographic elution, potentially affecting peak width and retention time, especially for tangeretin, where a 300 percent and 103 percent increase in peak intensity was obtained for mobile phases A and B, respectively, when mixtures with octan-1-ol were used. Due to co-elution between nobiletin and tetra-O-methyl scutelarein observed with mobile phase A, the method was validated using mobile phase B (acetonitrile and water 50:50 percent, v/v). Fluorescence detection provided lower LOD and LOQ values for sinensetin (1 and 3 microg mL -1 ) and nobiletin (13 and 44 microg mL -1 ) than reported in the literature. The method was applied to commercial sweet orange EOs samples, yielding consistent results for all PMFs.