Successful biological wastewater treatment depends on bacterial metabolic activity. Commercial fluorimeters are designed to monitor this activity using the native fluorescence of Nicotinamide Adenine Dinucleotide [NADH]. However, fluorescence measurements in wastewater treatment plants remain scarce due to difficulties with interpreting fluorescence data. This paper shows that fluorescence probe measurements taken from wastewater do not represent bacterial cell metabolic activity because intracellular NADH is likely swamped by the stable extracellular NADH fraction. Thus, a simple filtration/extraction/ centrifugation method was developed to collect the bacterial cells, extract the intracellular NADH using heat treatment in Tris buffer and collect the purified intracellular NADH fraction. NADH standards were used to quantify NADH from the unknown wastewater samples where limits of detection were between 1 nmolmL!1 and 0.35 mmolmL!1. Fluorescence of [NADH] greater than 0.35 mmolmL!1 was self-quenched. At high pH's NADH was stable outside the cell. NADH was stable at neutral and basic pH ranges of pH 7 to 11, but declined proportionally below a pH of 7. Since commercially available fluorescence probes used for measuring NADH are more likely detecting extracellular NADH, separating bacterial cells fromwater samples followed by NADH extraction was essential to distinguish intracellular and extracellular [NADH]. Here we have proposed three simple steps to meaningful measures of bacterial metabolic activity based on the autofluorescence of NADH. The three simple steps to getting it right are (1) filter the sample, (2) extract the bacteria collected on filter into a hot Tris buffer and (3) measure autofluorescence of the extract against a set of NADH standards. Future development of an on-line monitoring system based on these three steps is achievable with a little ingenuity