Over the centuries human have relied on plants for basic needs such as food, clothing, shelter and medicine. Despite the recent interest in drug discovery by molecular modeling, combinatorial chemistry, and other synthetic chemistry methods, naturalproduct-derived compounds are still proving to be an invaluable source of medicines for humans. The aim of the present study was to evaluate the In-Vitro Anticancer Screening of Whole plant of Eclipta alba Against HL-60 cell lines. In-Vitro anticancer screening of Ethanolic extract of whole plant of Eclipta alba claimed to be used traditionally in the treatment of various ailments including cancer against HL-60 lines by SRB was performed. It was observed from the study if GI 50 value of extracts is ? 20 µg/ml then it is considered to possess the anticancer activity. Here ethanolic extract of Eclipta alba showed result i.e., 30.5 µg/ml so it is considered as inactive on cancerous stem cells, but at the same time it was proved that a minimum of 40µg/ml concentration is sufficient to produce GI 50. We conclude that In vitro anticancer screening of ethanolic extract of whole plant of Eclipta alba against HL-60 cell lines by Sulphorhodamine (SRB) assay reveals that the plant having cytotoxic activity against the HL-60 cell lines and supports the folkloric usage of the plant. Preliminary phytochemical analysis of methanolic extract of methanolic extract of whole plant of Eclipta alba reveals the presence of steroids, flavonoids, glycosides, carbohydrates and tannins. The presence of above constituents in alone or in combination might be responsible for the observed anticancer activity