Raw data from four pooled Nigerian urine samples with iterative MS2 exclusion lists were processed for suspect screening to demonstrate the workflow's suitability for the suspect and nontargeted screening/analysis (NTS/NTA). On average, 14,126 and 18,408 features were picked in each sample in negative mode and positive mode, respectively. 16,288 negative features in 4,590 groups remained after removing features present in the blank and applying an intensity and replicate abundance filter. In positive ionization mode, 21,384 features in 5,937 groups remained after filtering. Features were included if they were present in at least 3 out of 4 replicates. Therefore the feature number after the filter application was higher than in the individual sample. A match with the suspect list was found for 706 (positive) and 749 (negative) peaks. The suspect list contained several isomers. Therefore in some cases, various molecules were suggested as an annotation. Altogether, 1,238 different compounds, partly observed in both ionization modes, were proposed as potential annotations for the feature groups. Identification levels 1-4 were established for 52% (370) and 58% (435) in positive and negative ionization mode, respectively (Table and). MS2 matches (level ≥ 3c) were obtained for 187 (positive mode) and 190 (negative mode) feature groups. The annotation of 377 feature groups in both ionization modes with an identification level of at least 3c was successful, consequently showcasing the workflow's ability to detect analytes without available reference standard and capture additional compounds potentially present in the samples.