Monitoring the quality of surface, industrial, waste and drinking water is a crucial aspect of environmental monitoring since the water quality directly influences the quality of life. An important part of water monitoring is the investigation of the bacterial load, which can be conducted by a variety of methods including enzyme assays of highly specific bacterial enzymes. The usage of artificial fluorogenic enzyme substrates (fluorognic probes) for these assays is now commonly in place in laboratories worldwide. However, traditional methods of identifying bacteria can be time consuming as they involve incubation times up to 72 hours. This high incubation time can be problematic as often fast definitive answers are required on environmental health risks. Furthermore, incubation methods can be misleading as not all bacterial strains are equally represented. In the last decade quantitative real time fluorescence (QRTF) assays have been developed without the need of incubation. However, this technology also requests more sophisticated fluorogenic enzyme substrates, as commonly used substrates limit the capabilities of QRTF measurements. Within this thesis, the problems of the most common commercially available substrate, 4-methylumbelliferyl--D-glucuronic acid during QRTF assays were analyzed. With the gained insight into the synthesis and purification of -glucuronidase substrates a variety of different fluorogenic substrates were designed and successfully applied in QRTF enzyme assays. In these novel substrates, self immolative linkers were implemented to access new fluorophores and fluorescence resonance energy transfer (FRET) mechanisms. Furthermore, with the separated reaction and measurement (SRM) device a new approach for QRTF assays was designed. In SRM assays the substrate is immobilized on a solid phase, after the enzymatic reaction the fluorophore is released and can be measured. Due to the separation of the substrate from the measurement unit, the background fluorescence is considerably diminished resulting in an increased sensitivity of the enzyme assay. In summary, this work shows novel sensitive and fast strategies for the investigation of the bacterial load of biological systems.