Healthcare settings present a challenging environment for assessing low-level concentrations of specific volatile organic compounds (VOCs) in the presence of high background concentrations of alcohol from the use of hand sanitizers and surface disinfectants. The purposes of this laboratory-based project were to develop and validate a sampling and analysis methodology for quantifying low-level VOC concentrations as well as high-level alcohol concentrations found together in healthcare settings. Sampling was conducted using evacuated canisters lined with fused silica. Gas chromatography/mass spectrometry analysis was performed using preconcentration (for ppb levels) and loop injection (for ppm levels). For a select list of 14 VOCs, bias, precision, and accuracy of both the preconcentration and loop injection methods were evaluated, as was analyte stability in evacuated canisters over 30 days. Using the preconcentration (ppb-level) method, all validation criteria were met for 13 of the 14 target analytes—ethanol, acetone, methylene chloride, hexane, chloroform, benzene, methyl methacrylate, toluene, ethylbenzene, m,p-xylene, o-xylene, alpha-pinene, and limonene. Using the loop injection (ppm-level) method, all validation criteria were met for each analyte. At ppm levels, alpha-pinene and limonene remained stable over 21 days, while the rest of the analytes were stable for 30 days. All analytes remained stable over 30 days at ppb levels. This sampling and analysis approach is a viable (i.e., accurate and stable) methodology that will enable development of VOC profiles for mixed exposures experienced by healthcare workers.