At present standard methods employed for the microbiological monitoring of bathing waters require at least 18 hours to perform and are based on culturing techniques. This is a huge drawback when immediate action is required. Real-time and on-line monitoring are key factors for consideration in current method development for continuous indicator organism detection in order to meet early warning requirements and water safety plans. Methods utilising β-D-Glucuronidase (GUD) activity as an indicator of Escherichia Coli presence use labelled glucuronides to produce optical signals. Fluorometric assays for the measurement of Escherichia Coli GUD activity are traditionally performed using the fluorogenic substrate 4-methyl-umbelliferone-β-D-glucuronide (4-MUG) which upon hydrolysis releases the fluorophore 4-methyl-umbelliferone (4-MU). The major drawback of 4-MU is its high pKa (7.8), which causes only partial dissociation at pHs around the optimum pH for GUD activity (6.5-7.0). To overcome this issue researchers have employed discontinuous enzyme assays which require the addition of alkali. In this context we explore the spectrophotometric properties of three fluorogenic substrates and their respective aglycons (Fig.1 ) for the continuous measurement of GUD activity and we apply the developed method for the rapid detection of Escherichia Coli in environmental water samples.