Highly quantitative metabolomics studies of complex biological mixtures are facilitated by the resolution enhancement afforded by 2D NMR spectra such as 2D 13C-1H HSQC spectra. Here, we describe a new public web server, COLMARq, for the semi-automated analysis of sets of 2D HSQC spectra of cohorts of samples. The workflow of COLMARq includes automated peak picking using the deep neural network DEEP Picker, quantitative cross-peak volume extraction by numerical fitting using Voigt Fitter, the matching of corresponding cross-peaks across cohorts of spectra, peak volume normalization between different spectra, database query for metabolite identification, and basic univariate and multivariate statistical analyses of the results. COLMARq allows the analysis of cross-peaks that belong to both known and unknown metabolites. After a user has uploaded cohorts of 2D 13C-1H HSQC and optionally 2D 1H-1H TOCSY spectra in their preferred format, all subsequent steps on the web server can be performed fully automatically, allowing manual editing if needed and the sessions can be saved for later use. The accuracy, versatility, and interactive nature of COLMARq enables quantitative metabolomics analysis, including biomarker identification, of a broad range of complex biological mixtures as is illustrated for cohorts of samples from bacterial cultures of Pseudomonas aeruginosa in both its biofilm and planktonic states.