A new method for the quantification of squalene in amaranth seed oil (ASO) is proposed. The spectroscopic techniques of ultraviolet–visible (UV–vis), fluorescence, and Fourier transform infrared (FTIR) were tested using partial least square (PLS) calibration models. Based on the results obtained from the reference chromatographic method, two ASO samples with the highest and lowest concentrations among totally 40 ASO samples were selected to prepare the calibration set (CS) (n = 20). All ASO samples were also kept as prediction set (PS) for the control of the predictive capacity of the chemometric model to be developed. To quantify the squalene compound in ASO, a totally of 26 PLS calibration models were tested using all spectroscopic techniques with various spectral pretreatments, and the best PLS model was selected by evaluating their statistical values of root mean square error of calibration (RMSEC), root mean square error of cross-validation (RMSECV), and R-square. The best calibration model was obtained from normal spectra in FTIR spectroscopy (4000–400 cm−1) with the lowest RMSEC of 0.0930, RMSECV of 0.1301, and the highest R-square of 0.9989 for calibration. The results of this study revealed that FTIR spectroscopy coupled with PLS regression could be used for fast, accurate, and environment-friendly quantification of squalene present in ASO samples.