Herein we report a multidisciplinary approach involving bioinformatics, chemical degradation, and precursor-directed biosynthesis to rigorously determine the absolute configuration of thailandamide A. On the basis of the stereochemical assignments, in silico predictions, and mutational pathway dissection, we also unveil the structure of the immediate, thermolabile PKS product thailandamide B. Elucidating the absolute configuration of complex polyketides with multiple chirality centers is often an ambitious undertaking which may require multidisciplinary approaches. In this work we describe a particularly challenging case of a stereochemical analysis to unravel the structure of a short-lived polyene, thailandamide A (1). We succeeded in the unequivocal determination of all six stereocenters of this fragile molecule (2R, 3S, 13S, 22R, 24R, 29R), which was only feasible by a combination of chemical degradation, derivatization, 1D/2D NMR analyses, and precursor-directed biosynthesis to yield the deoxy variant (6) of 1. The stereochemical elucidation in conjunction with complementary bioinformatic analyses implied the unexpected and quite unusual direct incorporation of d-Ala into the thailandamide backbone. Furthermore, PKS domain analyses (KR fingerprinting) guided us to the presumed immediate product of the tha PKS. Using a low temperature isolation protocol we indeed succeeded in isolating and fully characterizing the highly unstable thailandamide B (2). However, monitoring pathway dissection by HRMS and using an engineered DTE mutant eventually revealed that E/Z isomerization of the double bond at C16/17 occurs during polyketide chain elongation, likely catalyzed by the DH domain of split module 6. In sum, these unexpected results not only point out intriguing stereochemical aspects of polyketide biosynthesis, but also endorse the power of synergizing chemical and biological methods in natural product research.