To investigate the timing of epoxidation, aromatic ring formation, and the role of downstream epoxidase in the biosynthesis of lasalocid A (a polyether antibiotic from Streptomyces lasaliensis), we employed a chemical strategy using carba(dethia) mimics of malonyl units to intercept truncated biosynthetic intermediates from polyketide synthases (PKSs). Wild-type and engineered mutant strains (with deletions of epoxidase LasC/epoxide hydrolase LasB or point-mutated acyl carrier protein (ACP) domains) were grown in the presence of carba(dethia) N-acetyl cysteamine esters. Micro LC/HRMS analyses of bacterial extracts identified PKS-bound intermediates, including putative undecaketide dienes (8a–c), their oxidized counterparts (9a–c, 11a for ΔlasB mutants), and fully oxidized dodecaketide species (7a,b, 10a). These intermediates provide novel insights: epoxidation occurs on enzyme-bound substrates (likely ACP11/ACP10-bound precursors), aromatization may follow ether ring formation, and the epoxidase LasC is crucial for complete assembly of the polyketide backbone. This method of sampling PKS-bound intermediates in vivo advances understanding of lasalocid A ring formation mechanisms, particularly the relative timing of key biosynthetic steps and the role of downstream enzymes in polyketide processing.