Mid-infrared FTIR spectroscopy is an efficient tool for the monitoring of bioprocesses. The use of Fourier transform mid-infrared spectroscopy( FTIR) combined with PLS to predict the concentrations of glycerol and methanol in fed-batch cultivations of Pichia pastoris in a chemical medium was investigated by using the synthetic solutions as calibration set. Generally,all models were robust and performed well,the calibration model allowed standard errors of calibration( SEC) and standard errors of validation( SEV) for glycerol and methanol concentrations of 0. 171,0. 532,0. 129 and 0. 248 g/L,respectively. The results from 5 L Fed-batch experiments showed that FTIR data had a good agreement with control data,with standard error of prediction( SEP) of 1. 09 and 0. 86 g/L,respectively. Specific growth rate and specific production rate were compared between FTIR and DO-stat to control methanol concentration. Results showed that FTIR could limit the methanol concentration under 2%,which could maintain a higher specific growth rate and specific production rate that was effective to enhance the production.