A proteomic workflow for a simple loss-less manual nano-fractionation (300 nL/fraction) for low µg sample amounts which avoids the need to dry down or transfer fractions to autosampler vials is shown to be feasible. It is demonstrated that the conventional procedure of drying samples down followed by reconstitution negatively affects the number of protein and peptide identifications. Furthermore, these losses seem to disproportionately affect hydrophobic peptides from the drying down and reconstitution step. By collecting and concatenating the fractions while the outlet of the column is submerged in a small predefined volume of 0.2% formic acid, the content of acetonitrile in the collecting vials was lowered such that it was compatible with direct injection for the online analysis. This additionally resulted in a time gain of approx. an hour for the total fractionation time. Acetonitrile concentrations up to 7.5% do not seem to compromise the chromatographic performance in the online analysis. Using as little as 2 µg digested HeLa lysate, approx. 7000 protein groups could be easily identified with 2 or more unique peptides. This was the case when fractionation was performed at pH 10 as well as at pH 5.5.