Label-free quantification using liquid chromatography-mass spectrometry (LC-MS) has now become a widely accepted analytical approach for the comparison of differential protein expression levels across multiple samples. One major concern of current label-free strategies is the technical variability introduced at multiple points during sample preparation. Typical workflows require cell lysis with buffers containing detergents, overnight proteolysis, removal of potential interferences such as salts and detergents by solid phase extraction (SPE) and subsequent solvent evaporation and reconstitution prior to analysis. Each of these stages is likely to introduce sample variability. Here we present a new strategy, which incorporates an acid-cleavable detergent in the lysis buffer, one-hour digestion with a temperature stable, immobilized enzyme and no requirement for SPE clean-up. The entire sample preparation stage takes less than three hours from cell pellet to autosampler vial and sample handling is kept to an absolute minimum. Our data demonstrate a significant reduction in the technical variability of sample preparation compared to a typical protocol along with a dramatic time saving with no cost in terms of qualitative peptide identifications.