Twenty three 4-(2-amido-2-ethoxycarbonyl) ethylsulfenyl-4-deoxy-4'-demethylepipodophyllotoxin analogues were synthesized by three steps, in which trifluoroacetic acid was used as a condensation agent of 4'-demethylepipodophyllotoxin with L-cysteine ethyl ester hydrochloride without any protection of phenolic hydroxy and amino groups. All compounds were screened in vitro against L1210 leukemia and KB cells, in which, compounds 11, 16 and 18 exhibited more potent antitumor activity than etoposide.