Abstract: Untreated silicon targets compared to conventional stainless steel targets were used for MALDI analysis of small and large molecules. Samples and matrices were deposited on silicon wafers and commercial stainless targets and irradiated with a 337 nm N2 laser. The resulting ions were accelerated and mass separated in a time-of-flight mass spectrometer. The silicon target resulted in intense and reproducible signals. Peak resolution and S/N were not significantly affected by the substrate, but detection limit was approximately ten times better with silicon substrate. The most notable observations on silicon substrate were shot-to-shot and spot-to-spot reproducibility. The RSD of peak height and area for the silicon substrate is approximately 1.5 times better for shot-to-shot and 3 to 10 times better for spot-to-spot reproducibility compared to the stainless steel substrate.