Strigolactones (SLs) are a class of plant hormones regulating several aspects of plant growth and development according to nutrient availability, particularly the modulation of root and shoot architectures. Under nutrient deficiency, SLs are abundantly released into the soil to recruit a plant-beneficial partner, arbuscular mycorrhizal fungi (AMF), and establish plant-AMF symbiosis that provides the plant with minerals and water. However, released SLs are also seed germination signals for the root parasitic plants Orobanchacea family and pave their way to the host plants’ roots. “New comers” in the field of plant hormones, their large structural variety intrigues and led to ask why plants produce many different types of SLs. In this work, we generated tools that can help to link the SL structural diversity with their biological function(s). The most common way to evaluate SL activity is based on their ability to be parasitic seeds’ germination stimulants. Despite being the most sensitive assay for SL quantification, parasitic seed-based bioassays are laborious and time-consuming as performed usually manually. Therefore, we developed a detection algorithm, SeedQuant, which identifies and counts germinated and non-germinated seeds 600 times faster than a trained human; thus, reducing the data processing from days down to minutes. To gain quantitative insights in SL perception, depending on the structural diversity, we developed a precise and detailed protocol for the use of a genetically encoded biosensor in Arabidopsis protoplast, StrigoQuant. StrigoQuant takes advantage of the SL-dependent degradation of the repressor protein AtSMXL6 coupled with luciferase reporter proteins. We also tried to adapt this molecular sensor to the rice repressor protein D53, but the use of rice protoplasts made it very challenging. Moreover, to better understand the later steps in SL biosynthesis in vivo, we generated CRISPR/Cas9-based rice mutants and shed light on the biological function of different SLs at the organismal level. MAX1-900 mutants defined the minor role of the canonical SL 4-deoxyorobanchol (4DO) - a major plant SL - in determining rice architecture, while being a crucial contributor to rhizospheric interactions. Finally, we reviewed other strategies to decipher plant signaling pathways in general.