Previously, we reported that tasar silkworm pupal oil (TPO) is a rich source of alpha-linolenic acid (ALA) (37%). In this study, we have used three chromatographic techniques, namely preparative HPLC, gas chromatography-mass spectroscopy (GC-MS), and gas chromatography flame ionization detection (GC-FID), to purify, determine, and quantify alpha-linolenic acid (ALA), respectively, from TPO. Additionally, to determine the potential of the purified ALA as an anticancer drug, the sulforhodamine B (SRB) assay was utilised for in vitro investigation. Results revealed that ALA was successfully purified in a fraction of TPO by preparatory HPLC. It was discovered that only ALA was present in the purified fraction of ALA when it underwent GC-MS analysis, further confirming its purity. In addition, the quantification analysis using GC-FID showed that the concentration of ALA was 11760 ppm. Furthermore, anticancer analysis revealed purified ALA significantly inhibited the growth of a colon cancer cell line, (COLO-205) with a GI 50 ≤20. This is the initial investigation into isolating and purifying ALA from Tasar pupal oil. Keywords: Alpha-linolenic acid; GC-FID; HPLC; Omega-3Preparative, Tasar pupal oil.