Updated publication reference for PubMed record(s): 26862574. Proteomic workflows based on nanoLC-MS/MS data-dependent-acquisition analysis have progressed tremendously in recent years due to the technical improvement of mass spectrometers, and now allow to extensively characterize complex protein mixtures. High-resolution and fast sequencing instruments have enabled the use of label-free quantitative methods, which appear as an attractive way to analyze differential protein expression in complex biological samples. Classical label-free quantitative workflows are based either on spectral counting of MS/MS sequencing scans for each protein, or on the extraction of peptide ion peak area values in the LC-MS map composed of all the survey MS scans acquired during the chromatographic gradient. However, the computational processing of the data for label-free quantification still remains a challenge. Here, we provide a dual proteomic standard composed of an equimolar mixture of 48 human proteins (Sigma UPS1) spiked at different concentrations into a background of yeast cell lysate, that was used to benchmark several label-free quantitative workflows, involving different software packages developed in recent years. This experimental design allowed to finely assess their performances in terms of sensitivity and false discovery rate, by measuring the number of true and false-positive (respectively UPS1 or yeast background proteins found as differential). This dataset can also be used to benchmark other label-free workflows, adjust software parameter settings, improve algorithms for extraction of the quantitative metrics from raw MS data, or evaluate downstream statistical methods