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Visible and near-infrared (Vis/NIR) spectroscopy was investigated for fast and non-destructive determination of protein content in rapeseed leaves treated with herbicide of Pyribambenz-propyl (PP). 64 samples were used in the calibration set, whereas 32 samples in the validation set. Partial least squares (PLS) analysis was the calibration method as well as extraction method of latent variables (LVs). Certain selected LVs were used as the inputs of back propagation neural networks (BPNN) and least squares-support vector machine (LS-SVM). The prediction results demonstrated that LS-SVM outperformed PLS and BPNN methods. The correlation coefficient, RMSEP and bias in validation set by LS-SVM were 0.999, 59.562 and 7.437 for protein content, respectively. The results indicated that Vis/NIR spectroscopy combined with LS-SVM could be successfully applied for the detection of protein content of rapeseed leaves.