Abstract Mutational signatures connect characteristic mutational patterns in the genome with biological processes that take place in the tumor tissues. Analysis of mutational signatures can help elucidate tumor evolution, prognosis, and therapeutic strategies. Although tools for extracting mutational signatures de novo have been extensively benchmarked, a similar effort is lacking for tools that fit known mutational signatures to a given catalog of mutations. We fill this gap by comprehensively evaluating eleven signature fitting tools (well-established as well as recent) on synthetic input data. To create realistic input data, we use empirical signature weights in tumor tissue samples from the COSMIC database. The study design allows us to assess the effects of the number of mutations, type of cancer, and the catalog of reference signatures on the results obtained with various fitting tools. We find substantial performance differences between the evaluated tools. Averaged over 120,000 simulated mutational catalogs corresponding to eight different cancer types, SigProfilerSingleSample and SigProfilerAssignment perform best for small and large numbers of mutations per sample, respectively. We further show that ad hoc constraining the list of reference signatures is likely to produce inferior results and that noisy estimates of signature weights in samples with as few as 100 mutations can still be useful in downstream analysis.