Modern treatments of Parkinson's disease include the use of carbidopa and droxidopa along with levodopa that demonstrates the necessity for their binary and simultaneous determination in pharmaceutical tablets and biological matrices. This study presents a rapid, accurate, and precise method for the simultaneous determination of these analytes by liquid chromatography- tandem mass spectrometry method for the first time. Accordingly, performances of two types of analytical columns, namely, octadecylsilane and hydrophilic interaction liquid chromatographic columns, were tested and better resolution was obtained with the former. The pH of aqueous mobile phase (0.5% formic acid) was adjusted to 2.3 to ensure the analytes were in molecular form. Calibration graphs were obtained using multiple reaction mode and detection limits were in the region from 0.002 to 0.007 mu M for both columns. The method developed was applied for quality control samples and tablets and satisfactory recovery values from 100 to 106% were obtained.