Data-independent acquisition (DIA) methods aim to expand the benefits of low-throughput targeted proteomics to proteome-wide analyses. These methods rely on the use of several broadband isolation windows that select and fragment all peptide ions within a cycle. Isolation windows differ in that (I) they exhibit different widths, (ii) they are acquired either sequentially or in a non-consecutive order, and (iii) they are either juxtaposed or overlapped. Here we present DIA+, a novel DIA multiplexing scheme with isolation windows that combine signals from identical peptides with different charges. DIA+ is based on the co-isolation of charge +2 and +3 precursor ions from identical peptide sequences to combine their signal and therefore, increase the number of MS2 peptide fragment ions detected. DIA+ combines three non-consecutive m/z ranges, based on the mass difference between charge +2 and +3 peptides, into a composite 24 Da isolation window. Forty of these combined 24 Da windows cover a range of 400-1350 m/z. The performance of DIA+ was compared with other reference methods in the field, and with a DIA+ control method in which each 24 Da isolation window results from the combination of three consecutive m/z ranges. The combination of improved signal-to-noise and sequence coverage increases the confidence on peptide identification and results in a significant increase in the number of identified peptides and proteins.