A novel headspace gas chromatography-mass spectrometry (HS-GC/MS) method for identifying and quantifying 3,3-dimethyl-1-butanol (DMB), a bioactive compound, in extra virgin olive oil was developed. In this study, solvents with similar properties to DΜB but potentially different molecular masses, namely isopropanol, n-butanol, n-pentanol, and 2-pentanol, were tested and 2-pentanol was selected as the internal standard due to both chromatographic separation and distinctive mass spectrum pattern. The method for DMB determination and quantification was validated according to Eurochem CITAC Guide. Sensitivity, specificity, linearity, accuracy, and precision parameters were evaluated. The validation process included an assessment of the method’s robustness and repeatability, ensuring that it produces reliable results for future analyses of olive oil. A linear regression equation was developed for DMB concentrations ranging from 100 to 5,000 µg/L, expressed as y = x.11.462.10⁻⁶, where y represents the peak area ratio and x is the DMB concentration in µg/L. This equation has a high correlation coefficient of 0.9989 (R²), and the uncertainty budget was estimated to be 8.22% at a confidence level of k = 2. In addition, the lower concentration range of 0 to 100 µg/L was characterized by the equation y = x.17.860.10⁻⁶ which has a correlation coefficient of 0.9983 (R²). The uncertainty of DMB measurements in extra virgin olive oil was estimated to be 7.0% at a confidence level of k = 2. This new method was applied to determine the presence of DMB in various olive oil samples from Greece and Türkiye. The DMB values for olive oil samples were: N1(Plus health blue multi varietal) 9.7 μg/L, Ν2 (Plus health DMB multi varietal) 11.4 μg/L, N3 (Fyllikon first harvest organic) 8.3 μg/L, N4 (Plus health green multi varietal) 7.8 μg/L, N5 (Agourelaio early harvest organic) 6.8 μg/L, N6 (unknown) 4.8 μg/L, N7 (Armonia monovarietal organic) 4.8 μg/L and N8 (Edremit type olive oil from Kırkağaç, Manisa) 8.8 μg/L and N9 (Market product in Türkiye) 1.5 μg/L.