The in vitro micropropagation of the seeds of Tanacetum parthenium (L.) Schultz-Bip. family Asteraceae was performed on half strength Murashige and Skoog (MS) medium containing 0.2% gibrellic acid. The explants were in vitro cultured on MS-medium using different plant growth regulators, culture media ingredients and carbon sources. Parthenolide content in the different established cultures was studied and compared with that present in the open field herb. Results revealed that using half strength MSmedium containing 50 g/l glucose or fructose as a carbon source and 0.5 mg/l benzyl aminopurine (BAP) resulted in significantly higher parthenolide content than that present in the open field herb. In addition, bud sprouting ability, number and length of shootlets and number of leaves of different explants under different treatments were studied. The in vitro callus formation was conducted on MSmedium using different plant growth regulators and culture media ingredients. Parthenolide was detected in callus culture for the first time in only two different hormonal treatments which were MS medium containing 0.5 mg/l BAP or 0.5 mg/l naphthalene acetic acid (NAA). In addition, callusing capacity and weight of callus of different explants under different treatments were also determined. Parthenolide content was determined in the explants as well as in callus using RP-HPLC on Luna C18 column and SPD-10A UV detector. The mobile phase used was acetonitrile: water (55 : 45) and the flow rate was 1.5 ml/min at the ambient temperature.