Advantages of dried blood spot include low invasiveness, ease and low cost of sample collection, transport, and storage. We used tandem mass spectrometry (LC-MS/MS) to determine phenobarbital levels on dried blood spot specimens and compared this methodology to commercially available particle enhanced turbidimetric inhibition immunoassay (PETINIA) in plasma/serum samples. The calibration curve in matrix using D(5)-phenobarbital as internal standard was linear in the phenobarbital concentration range of 1-100 mg/L (correlation coefficient 0.9996). The coefficients of variation in blood spots ranged 2.29-6.71% and the accuracy ranged 96.54-103.87%. There were no significant differences between the concentrations measured using PETINA and LC-MS/MS (both had similar precision and accuracy) however, LC-MS/MS allows at least 1.5 times higher throughput of phenobarbital analysis and additionally offers ease of sample collection which is particularly important for newborns or small infants.