Direct alcohol biomarkers are of growing interest for the assessment of alcohol consumption, with particular interest in phosphatidylethanol (PEth) in recent years. PEth is only formed when alcohol is present in the body. However, there is no statement about how much the PEth concentration increases after single moderate alcohol consumption. This study was conducted to determine the increase in PEth concentrations after a single drinking event. Additionally, a new volumetric sampling device (DBSV) was evaluated, which was designed to simplify further sampling processes and to allow for easy self-sampling. Dried blood samples from 31 volunteers were collected before and after single alcohol consumption with mean maximum breath alcohol concentration (BrAC) of 0.4 mg/L (range: 0.30 - 0.55 mg/L). PEth concentrations were determined after automated extraction by liquid chromatography-tandem mass spectrometry. PEth 16:0/18:1 and PEth 16:0/18:2 concentrations increased to an average of 45 ng/mL each in patients starting below 20 ng/mL (range: 25.0 – 57.0 ng/mL for PEth 16:0/18:1; range 26.8 – 62.3 ng/mL for PEth 16:0/18:2). PEth concentrations in patients starting above 20 ng/mL increased by a mean of 30 ng/mL (range: 6.2 - 71.3 ng/mL for PEth 16:0/18:1; range 8.8 - 65.3 ng/mL for PEth 16:0/18:2). In addition, the comparison of the new sampling device DBSV with a standard filter paper card (with volumetrically applied 20 µL blood samples) yielded a close agreement for the determined PEth concentrations in 24 forensic samples and 3 external controls. Therefore, the sampling device DBSV proved to be suitable for the determination of PEth concentrations in blood.