Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is an emerging technique for the determination of the molecular weight of biomolecules and their non-covalent complexes without fragmentation. One problem with this technique is the use of excess amounts of matrices, which may produce intense fragment ions and/or clusters at low mass ranges between 1 and 800 Da. These fragments lead to interference, especially concerning the signals of small target molecules. Here, a simple, reusable, and quite inexpensive approach was demonstrated to improve the effectiveness of laser desorption/ionization mass spectrometry (LDI-MS) analysis, especially for small molecules, without using matrix molecules. In this study, substrates with controllable morphologies and thicknesses were developed based on the self-assembly of silane molecules on silicon surfaces using N-(3-trimethoxysilylpropyl)diethylenetriamine (TPDA) and octadecyltrichlorosilane (OTS) molecules. Prepared substrates with nano-overlayers were successfully used in the analysis of different types of small target molecules, namely acrivastine, L-histidine, L-valine, L-phenylalanine, L-arginine, L-methionine and angiotensin I. Our substrates exhibited clear peaks almost without fragmentation for all target molecules, suggesting that these surfaces provide a number of important advantages for LDI-MS analysis, such as ease of preparation, costs, reusability, robustness, easy handling and preventing fragmentation. (C) 2012 Elsevier B.V. All rights reserved.