The interest in two-dimensional liquid chromatography separations is growing every year together with the number of open questions on the benefits of multidimensional systems in comparison to one-dimensional liquid chromatography. In order to solve some of these open questions this work presents a comparison of one-dimensional and microscale two-dimensional liquid chromatography coupled to high resolution mass spectrometry for targeted analysis in wastewater. The comparison is based on the evaluation of a reference standard mixture containing 99 compounds and a real wastewater sample. For the evaluation and compound identification three different criteria were chosen. At first, a deviation of ±5 ppm from the exact mass was defined as acceptable to include the compound for further evaluation. To eliminate false positive results, a maximum retention time deviation of less than 2.5% for each compound of the reference standard and the compounds detected in the wastewater sample was defined for a positive identification as a second criterion for 1D-LC and the second dimension of 2D-LC. In the third step, fragment information from MS/MS experiments was used for further identification of compounds in wastewater. Additionally, the influence of a higher mass accuracy of 1 ppm on the number of identified compounds in comparison to a mass accuracy of 5 ppm was investigated. The results showed that the number of identified compounds was higher by a factor of three in the wastewater sample when using the microscale 2D-LC approach. Moreover, a higher reliability for compound identification is obtained when using retention time and MS/MS information as identification criteria instead of only applying high mass accuracy of 1 or 5 ppm.