A remarkably concise seven- to eight-step total synthesis of a systematic series of key vinblastine derivatives is detailed and used to characterize the importance and probe the role of the C5 ethyl substituent (R = H, Me, Pr, CH=CH2, C CH, CH2OH, and CHO vs Et). The analogues, which bear deep-seated structural changes accessible only by total synthesis, were prepared using a powerful intramolecular [4 + 2]/[3 + 2] cycloaddition cascade of 1,3,4-oxadiazoles ideally suited for use in the assemblage of the vindoline-derived lower subunit followed by their incorporation into the vinblastine analogues through the use of a single-step biomimetic coupling with catharanthine. The evaluation of the series revealed that the tubulin binding site surrounding this C5 substituent is exquisitely sensitive to the presence (Et > H, 10-fold), size (Me <= Et > Pr, 10-fold), shape (Et > CH=CH2 and C CH, >4-fold), and polarity (Et > CHO > CH2OH, >10-20-fold) of this substituent and that on selected occasions only a C5 methyl group may provide analogues that approach the activity observed with the naturally occurring C5 ethyl group.