The medicines prepared by using exudates of Commiphora mukul (Stocks) Hook. are described in Ayurveda under Guggula Kalpana which are among such valuable dosage forms. According to retrospective literary review, the combination of Kukkutnaki (Aspidium cicutarium Sw.) and purified Guggula(Commiphora mukul (Stocks) Hook.) was first mentioned in the book Chikitsa pradeep named as Kukkutnaki Guggula. Since last 3 decades, it was documented as an herbal drug which is used for cysts, goiter, tumors, tonsillitis, abscess, mansvaha strotas ailments, which are burning issues worldwide. Due to its observed clinical efficacy in Arbuda (~Cancer), the current in-vitro anticancer study was conducted with an aim to check its anticancer effect on human hepatoma cell line-HEPG2 of Liver; PC-3 and DU145 cancer cell lines of Prostate; Ovcar-3, A2780, SK-OV-3, PA-1 cancer cell lines of Ovary and ACHN renal cancer cell line of Kidney. The current in vitro study was conducted at ACTREC, Kharghar, Navi Mumbai. The selected cancer cell lines were procured from ATCC, USA and NCCS Pune. The Sulforhodamine B (SRB) Assay protocol was followed to observe the activity of the study drug. The positive control was Adriamycin in the study. The growth curve graphs were plotted and LC50, GI50, TGI values were calculated. Kukkutnakhi Guggula was found safe for oral administration, non- toxic at cellular level (LC50 values were > 160) and have moderate activity on HEPG2, Ovcar-3, DU145, ACHN cancer cell lines and had shown negligible activity on A2780, SK-OV-3, PA-1 and PC-3 cell lines. This work provides scope to study its effect on targeted cancers, specific in vivo scientific studies and human clinical trials for further researchers.