We recently showed that electrowetting-enhanced sample preparation for MALDI-MS (eMALDI) can increase the intensity of the MALDI signal by 2-25 times compared to conventional drop drying by concentrating all the dried sample in a single spot rather than leaving behind a heterogeneous coffee stain pattern. Here, we demonstrate that the eMALDI signal enhancement can be further increased to more than 100x by systematically optimizing the electrowetting actuation frequency and amplitude. This enables 30x signal increase for a peptide standard. Simultaneously drop drying times can be reduced approximately five times by increasing the actuation voltage and/or decreasing the initial drop volume.@en