Although measurement of chemiluminescence has become a widespread tool in the study of phagocytosis of peripheral neutrophils, several problems linked to spontaneous fluctuation in chemiluminescence and the number of variables involved have occasionally either limited its usefulness for clinical and experimental purposes or compelled operators to take particular care when using the technique. In the present paper, sources of variability are investigated and most of the parameters involved are thoroughly analysed and step-by-step normalised. A stochastic calibration procedure for validation of the method is applied and a monofunctional test protocol for quantitative evaluation of plasma opsonizing activity in whole blood chemiluminescence is suggested. With regard to the goal of proposing a reverse monofunctional test, we discuss the reasons why further studies aimed at standardised evaluation of the cellular components are needed.