The synthesis and cytotoxic studies of a new series of 16E-arylidene androstene derivatives are reported herein. The impact of incorporating bis-tertiary amino functionalities in the steroid skeleton on cytotoxicity has also been observed. The compounds have been evaluated at National cancer Institute, Bethesda, Maryland, USA for their antineoplastic activity against various tumor cell lines. The synthesized 16E-arylidenosteroids exhibited significant cytotoxicity. Bis-tertiary amino steroid 29 possessing a diethylaminoalkoxy functionality was the most promising compound of the series with a total IP and SC score of 20 in in vivo hollow fiber assay and was selected for further detailed in vivo xenograft testing.