The study aims at the isolation of gentiopicroside (GPC) from Gentiana lutea L. root extracts. GPC is a bioactive glycosylated secoiridoid from Gentiana spp. (Gentianaceae) proved to exhibit broad pharmaceutical activities, including anti-inflammatory, hepatoprotective and spasmolytic action. Due to its marked potential, an efficient recovery method of high purity GPC from G. lutea roots was elaborated using the combination of Centrifugal Partition Extraction (CPE) that offers the advantage of industrial scalability, and Preparative High Performance Liquid Chromatography (prep-HPLC) techniques. For this purpose the root hydroalcoholic extract of G. lutea was initially fractionated in a 0.3 L volume CPE column using the biphasic solvent system that was composed of heptane: ethyl acetate: ethanol: water 1:8:3:5 (v/v/v/v) in elution-extrusion mode. As a result, 2.2 g of GPC (purity > 90%) and 0.77 g of GPC-enriched fractions (purity > 80%) were obtained from 31.5 g of the extract within 6 h (5 consecutive CPE fractionations). The following prep-HPLC based purification step increased the quantity and purity of the finally isolated secoiridoid to 2.21 g of > 96% pure GPC. The successful recovery of large amounts of pure GPC enabled the study on the anti-proliferative activity against the following cancer and normal cell lines: lung (A549, NCI-H1299), breast (MCF7, MDA-MB-468), glioblastoma (T98G), rhabdomyosarcoma (TE671), colon (HT-29, DLD-1) cancer cells and normal colon epithelial cells (CCD 841 CoTr). The MTT assay enabled the evaluation of the dose-dependent growth inhibition effect that was evident in all analyzed cancer cell lines, however, TE671 cells were the most sensitive (IC50 = 0.078 mg/mL) and MDA-MB-468 cells were the least sensitive (IC50 = 0.208 mg/mL) to the GPC treatment. The tested compound showed only a weak cytotoxic effect against normal cells (CCD 841 CoTr)