Comprehensive drug-screening performed by liquid chromatography−high resolution mass spectrometry (LC−HRMS) enables identification of hundreds to thousands of drug compounds in a single analysis. Forensic drug screening is generally performed with positive electrospray ionization (ESI⁺), targeting basic drugs; however, a few toxicologically important drugs such as barbiturates, may require analysis by negative ESI. In this work, screening targets for barbiturates were determined using our LC−HRMS screening with ESI⁺. For several years, our forensic whole blood samples have been analyzed using the LC−HRMS−ESI⁺ screening in parallel with a multi-target LC–MS/MS−ESI⁻ method. From 2014 to 2018, 23 samples were positive for phenobarbital (0.5−81 mg/kg). Retrospective data analysis of 4816 blood samples (15 positive) revealed several potential screening targets for phenobarbital. The targets were tentatively identified by exact mass and isotopic pattern as uncommon adducts of phenobarbital and as a decomposition product of phenobarbital N-glucoside (C₁₇H₂₄N₂O₇). Analysis of a test set containing eight positive (0.5–65 mg/kg phenobarbital) and 31 negative samples supported the use of the observed target m/z 323.0614 at 5.14 minutes, corresponding to the [M + HCOONa+Na]⁺ adduct of phenobarbital. The [M + HCOONa+Na]⁺ adduct was confirmed as a screening target for common barbiturates, by analysis of barbiturate reference standards in ESI⁺/ESI⁻. The [M + HCOONa+Na]⁺ adduct allowed retrospective analysis with 91% sensitivity (n = 23) and 100% specificity (n = 4855) for phenobarbital in our existing LC−HRMS−ESI⁺ screening. The two negative results were the two whole-blood samples with the lowest phenobarbital concentration (<1.8 mg/kg). Thus, a specialized screening is not necessary and use of this adduct likely enables screening for other barbiturates.