A matrix-assisted laser desorption ionization time-of-flight mass spectrometry(MALDI-TOF MS) method was set up for simultaneously screening and analysis of 3 kinds of anti-hypertensive substances which is clonidine hydrochloride (HCD)， atenolol (ANL) and reserpine (RSP) in dietary supplements. The different spotting approaches and laser power intensities of 3 kinds of anti-hypertensive substances were compared， and the methodology validation experiments were also completed. As a result， α-cyano-4-hydroxycinnamic acid (HCCA) was used as matrix， and samples were spotted by mixed approach under the reflect linear positive mode and laser power of 60%. Under the optinal condition of MALDI-TOF MS， the 3 objectives could obtain stable signals， high intensities and well-repeated mass spectrometrics. The results of method validation showed that the linear relationship was excellent among 10 to 100 ng/mL， and the regression coefficients (r) were all above 0.99. The limit of identification (LOI) were between 0.005 to 0.25 μg/g， and the recoveries were in the range of 67.56%-104.70% among liquid， solid and half-solid dietary supplements samples， while the relative standard deviations (RSDs) were ranged from 0.43% to 10.51% (n=3). This method occupied with high sensitivity， accuracy precision， anti-interference ability， low chemical solvents and environmental friendly， which could satisfy multitudinous batches of dietary supplement samples. Meanwhile， this method filled a gap of traditional matrix used in MALDI-TOF MS to screen and analyze small molecule compounds in dietary supplements.