Recent progresses in the development of fluorescent technologies become a reliable device for drug discovery research. The fluorescence tools offer attractive options for an opportunity to visualize the effects of drug candidates in the cells. The fluorescent tools, such as fluorescent protein, are regularly used in a range of drug discovery processes. A better understanding and use of fluorescent technologies facilitate drug discovery research faster and can open up new applications. Therefore, we have provided information about some new generation fluorescent reagents (GFP and fluorophores). This review illustrates how fluorescent technologies and fluorescent tools are contributing to the drug discovery process mainly high-throughput screening (HTS), disease mechanism based target discovery, disease-genes-based target discovery, ‘target classes’ based target candidate discovery, physiology-based drug discovery, genomics-based drug discovery, target validation and their future perspectives.