Abstract Background Strigolactones represent the most recently described group of plant hormones involved in many aspects of plant growth regulation. Simultaneously, root exuded strigolactones mediate rhizosphere signaling towards beneficial arbuscular mycorrhizal fungi, but also attract parasitic plants. The seed germination of parasitic plants induced by host strigolactones leads to serious agricultural problems worldwide. More insight in these signaling molecules is hampered by their extremely low concentrations in complex soil and plant tissue matrices, as well as their instability. So far, the combination of tailored isolation—that would replace current unspecific, time-consuming and labour-intensive processing of large samples—and a highly sensitive method for the simultaneous profiling of a broad spectrum of strigolactones has not been reported. Results Depending on the sample matrix, two different strategies for the rapid extraction of the seven structurally similar strigolactones and highly efficient single-step pre-concentration on polymeric RP SPE sorbent were developed and validated. Compared to conventional methods, controlled temperature during the extraction and the addition of an organic modifier (acetonitrile, acetone) to the extraction solvent helped to tailor strigolactone isolation from low initial amounts of root tissue (150 mg fresh weight, FW) and root exudate (20 ml), which improved both strigolactone stability and sample purity. We have designed an efficient UHPLC separation with sensitive MS/MS detection for simultaneous analysis of seven natural strigolactones including their biosynthetic precursors—carlactone and carlactonoic acid. In combination with the optimized UHPLC–MS/MS method, attomolar detection limits were achieved. The new method allowed successful profiling of seven strigolactones in small exudate and root tissue samples of four different agriculturally important plant species—sorghum, rice, pea and tomato. Conclusion The established method provides efficient strigolactone extraction with aqueous mixtures of less nucleophilic organic solvents from small root tissue and root exudate samples, in combination with rapid single-step pre-concentration. This method improves strigolactone stability and eliminates the co-extraction and signal of matrix-associated contaminants during the final UHPLC–MS/MS analysis with an electrospray interface, which dramatically increases the overall sensitivity of the analysis. We show that the method can be applied to a variety of plant species.