Abstract: A reversed-phase HPLC method was developed for simultaneous quantification of 6 plastid pigments in tobacco sample.The extraction solvent,extraction mode,extraction time,mobile phase,column temperature and flow rate were tested and optimized.The plastid pigments in 10 tobacco samples were quantitatively determined,the results showed that:1) the plastid pigments in tobacco were satisfactorily extracted with 90% acetone aqueous solution under ultrasonic for 20 minutes;2)6 plastid pigments were completely separated by reversed-phase HPLC with a Waters Nova-Pak-C₁₈ column under a gradient elution with isopropyl and acetonitrile-water(v:v=80:20) as mobile phase at a flow-rate of 0.5 mL/min;3) the recoveries of pigments were from 96.7% to 110.7% with the RSD of under 5%;4)6 plastid pigments,including neoxanthin,violaxanthin,lutein,chlorophyll-b,chlorophyll-a,and β-carotene,were detectable and with relatively higher content in 2 samples of freeze-dried fresh tobacco,while only lutein and β-carotene were found in cured tobacco and cigarettes,and moreover,at far lower concentration comparing with fresh tobacco.This method is suitable for determining the 6 plastid pigments in batches of tobacco samples.