Abstract Proteins destined for the secretory pathway start their journey in the endoplasmic reticulum and transit through the Golgi complex to be delivered to their destination compartment. Over the last decades, several fluorescence-based assays were developed to analyze the transport of proteins along the secretory pathway. In this review, we briefly introduce the existing tools. We provide detailed protocols to allow the reader to use the newly developed secretory assay termed the RUSH system (Retention Using Selective Hooks). This assay enables the synchronous release of one to three cargos of interest from a donor compartment (the endoplasmic reticulum). Analysis of the transport steps of the cargos from the donor compartment to the acceptor compartment is accomplished by fluorescence-based methods.