Attenuated total reflectance-Fourier transform infrared (ATR-FTIR), ultraviolet–visible (UV–Vis), and fluorescence (FL) spectroscopic techniques, combined with partial least-square (PLS) regression with various spectral derivatization methods were tested for the quantitative determination of squalene content of extra virgin olive oils (EVOOs). A set of 90 calibration standards covering a wide range of squalene (3.25–12.54 mg/kg) was used to build up the calibration models. The root mean square error of calibration (RMSEC), the root mean square error of cross-validation (RMSECV), and the root mean square error of prediction (RMSEP) were calculated for evaluation of 18 different calibration models. 50 different brands of EVOOs, which are also analyzed in terms of quality indexes, fatty acid composition, and squalene concentration (3.25–12.54 g/kg) were used for checking the predictive capacities of the calibration models. The best predictions were achieved using normal spectra in FL spectroscopy with the lowest RMSEC of 0.1065, RMSEV of 0.1310, and RMSEP of 0.1500 in the spectral region 250–730 nm. Thus, FL spectroscopy combined with PLS regression is proposed as a fast, accurate, and environmentally friendly approach that can be efficiently used in determining squalene in EVOOs.