An automated reverse flow injection analysis (r-FIA) system using stop-flow technique for quantifying methanol based on the enzymatic reactions of alcohol oxidase and peroxidase was developed. The system permitted methanol analysis in a linear range of 0.006-0.1 g methanol l(-1) without external dilution, and with a sampling frequency of 12 analyses per hour, with a relative standard deviation of 1.16%. The analyser was validated analysing samples from a Pichia pastoris fermentation producing a heterologous protein.