The CH2Cl2-MeOH extract of the Mediterranean sponge Crambe crambe was investigated via UHPLC-HRMS/MS employing manual dereplication and in silico mass spectrometry tools. A deconvolution approach was implemented for the extensive metabolic characterization of the sample, resulting in the annotation of 53 compounds. The analysis of data-dependent HRMS/MS scans was conducted to establish fragmentation patterns characteristic of each crambescin A, B, and C sub-families. Among the 39 compounds identified from these groups, 22 analogues were reported for the first time including 4 new homologous series that differed by the ratio of methylene units in the upper (n + 2) and lower (m + 2) alkyl side chains. More specifically, crambescins presenting m = 5 or 6 and n = 5 (compounds 7, 11, 22 and 24) as well as m = 5 or 6 and n = 4 (compounds 5, 6, 8, 9, 12 and 14) were characterized. Additionally, four new features, potentially corresponding to new crambescidin analogues (compounds 13, 15, 35, and 39), were also reported. The identity of the dereplicated features was further validated by studying crambescins' spectral similarities through a feature-based molecular networking approach. Overall, this study suggests UHPLC-HRMS/MS-through the integration of manual and computational dereplication approaches-as a valuable tool for the investigation and high-throughput characterization of the C. crambe metabolome.