Tryptophan is an essential amino acid that plays an important role in cell metabolism, and kynurenine is its main metabolic pathway. By using ultra-high-performance liquid chromatography coupled to electrospray ionization triple-quadrupole mass spectrometry, tryptophan and kynurenine were determined using amlodipine as an internal standard. The analysis was carried out on an ACE-C18 (4.6 mm × 50 mm, 5 μm) reversed-phase analytical column using the gradient elution mode. For quantitative determination, amlodipine was used as an internal standard. Detection was performed using multiple reaction monitoring in electrospray ionization mode at m/z 205.1 → 117.7 and 187.9 for tryptophan, m/z 209.1 → 146 and 93.9 for kynurenine, and m/z 409.2 → 294.1 for the internal standard. Good linearity of the analyte to internal standard peak area ratios was seen in the concentration range 1.25-4000 ng/mL for tryptophan and 0.5-1600 ng/mL for kynurenine. The method showed excellent linearity with regression coefficients of 0.99 for kynurenine and 0.996 for tryptophan. The limits of quantification were 0.55 ng/mL for tryptophan and 0.47 ng/mL for kynurenine. The % RSD for all analytes ranged from 0.3 to 3.4% for intraday and 0.4 to 8.9% for interday experiments. A simple LC-MS/MS method has been developed and validated for measuring Kyn and Trp by using an affordable and more easily available internal standard, which is amlodipine.