Abstract: A flow injection immunoassay (FIIA) using ortho-phthalaldehyde (OPA) derivatization was developed for online monitoring of protein products in biotechnology processes. Recombinant tissue-type plasminogen activator (rt-PA) and its antibodies were used in these studies as a model system. The derivatization of rt-PA with OPA led to a lower detection limit (higher fluorescence intensity) and an increase of sensitivity (higher slope of calibration curve) for rt-PA compared with the method without OPA derivatization. The derivatization of rt-PA with OPA was also systematically characterized: influence of pH, derivatization time, OPA concentration. and sample volume on fluorescence intensity. Interferences caused by proteins were investigated with different washing buffer solutions A 0.1 M potassium phosphate buffer (PPB) solution containing 1 g L-1 polyethylene glycol (PEG) was found to be optimal for minimizing the interference with BSA in the measurement. Online monitoring for rt-PA is carried out on a simulated bioprocess in the range 0-20 µg mL-1. The data obtained showed good agreement with the conventional off-line enzyme linked immunosorbent assay (ELISA) data. The FIIA using o-phthalaldehyde (OPA) derivatization is a valuable tool for online monitoring of low concentration protein products in biotechnology production processes.