Nardostachyos Radix et Rhizoma (NR), the root and rhizome from either Nardostachys jatamansi Batal or Nardostachys jatamansi DC, is known to have biological functions including neuro-protective and anti-pancreatitis activity. The main bioactive compounds within NR are all classified as sesquiterpenes, and include desoxo-narchinol A, nardosinonediol, and nardosinone. Although NR is a valuable herb that is widely used in many Asian countries, robust quality control protocols for NR are still in question, especially those that can analyze the three main active compounds. Current quantitative methods within the Chinese Pharmacopoeic use nardosinone as a marker compounds. One compound cannot represent a complicated matrix, and is thus insufficient to control the quality of this herbal medicine. Moreover, there are no high-performance liquid chromatography (HPLC) methods that can simultaneously analyze desoxo-narchinol A (DA), nardosinonediol (NE), and nardosinone (ND) within NR. This study aimed to establish an efficient quality control protocol by developing an analytical method that simultaneously detects the three sesquiterpenes with HPLC using response surface methodology (RSM) to optimize sample preparation. Optimized HPLC conditions included a mobile phase of 0.1% formic acid in water (A), and a 0.1% formic acid in acetonitrile (B) under an elution program of 20% B–80% B for 30 min at 254 nm. The method was well validated, demonstrating satisfactory linearity, accuracy, precision, recovery, repeatability, and stability. Optimized conditions for creating the analytical sample were predicted by RSM using a Box-Behnken design. These conditions included reflux at 70 °C for 3 h using 24.98% ethanol as the extraction solvent (solvent: solid ratio = 78.81 mL/g). The relationship between the results between predicted and experimental conditions was well correlated, and varied between 96.48%–102.11%. Thus, our developed HPLC method, paired with optimized sample preparation conditions, accurately quantified all three sesquiterpenes, and may thus be a prospective means of controlling the quality of NR.