Marine dinoflagellates produce a unique family of bioactive substances featuring multiple ether rings aligned in a ladder shape. These are large, complex molecules with potent bioactivity. Targeted chiral centers sit on either the skeletal ladders or on the side chains of these compounds. However, the laborious steps of isolation and purification severely diminish the amount of sample available for assigning these chiral centers via structural investigations. Three important methods were used to assign the stereochemistry of the molecules, (a) circular dichroism (CD) spectroscopy, (b) labeling with fluorescent chiral reagents for high-performance liquid chromatography (HPLC) analysis, and (c) derivatization with anisotropic reagents for nuclear magnetic resonance (NMR) analysis. The addition of fluorescent chiral reagents allowed for the use of much less material than typically required. In this review, we present examples of the determination of absolute configurations in ladder-shaped polyethers. The targeted compounds include ciguatoxins (CTXs), gymnocin-B, gambieric acids, prymnesin-2, maitotoxin, yessotoxins, gambierol, brevisamide, and brevisin.