Simultaneous exposure of Au and In2O3/SnO2 (ITO) electrodes to an equimolar solution of a thiol and a carboxylic acid or a thiol and phosphonic acid results in the selective attachment of the thiol to the Au electrode and the carboxylic or phosphonic acid to the ITO electrode. This selective surface-attachment chemistry is termed “orthogonal self-assembly’’ (OSA) and can be used to direct the spontaneous assembly of molecular reagents onto Au and ITO microstructures. The selectivity of the thiols for Au and the carboxylic or phosphonic acids for ITO is determined by a combination of cyclic voltammetry experiments using ferrocene-tagged molecules, scanning Auger microscopy, and imaging secondary ion mass spectrometry (SIMS) to map the distribution of thiols, carboxylic acids, and phosphonic acids on derivatized Au and ITO microstructures. Simultaneous exposure of Au and ITO electrodes for 30 min to an equimolar solution of 11-mercaptoundecanoylferrocene (I) and 12-ferrocenyldodecanoic acid (III) results in a coverage ratio I:III of approximately 100:1 on Au and 1:45 on ITO, as determined by cyclic voltammetry. A 30-min exposure of Au and ITO electrodes to an equimolar solution of I and 6-ferrocenylhexylphosphonic acid (V) yields a coverage ratio of I:V of 30:1 on Au and better than 1:100 on ITO. The coverages of I, III, and V on the Au and ITO electrodes can be determined using cyclic voltammetry by virtue of the difference in redox potential between the acylferrocene center in I and the alkylferrocene centers in III and V. Typical converages of I on Au (4 × 10–10 mol/cm2) and III and V on ITO (6 × 10–10 and 3 × 10–10 mol/cm2, respectively) after 30 min of derivatization correspond to approximately a monolayer of redox-active molecules in each case. Long derivatization times (12–15 h) result in small or insignificant changes in the coverage ratios of these reagents on both Au and ITO electrodes, demonstrating that the OSA is essentially complete within 30 min. Surface analysis by X-ray photoelectron spectroscopy, scanning Auger microscopy, and imaging SIMS of Au and ITO microstructures and Si3N4 surfaces exposed to equimolar solutions of I and perfluorodecanoic acid (IV), or 12,12,12-trifluorododecanethiol (II) and V, reveal the selective assembly of these reagents on the Au and ITO microstructures and their absence on the insulating Si3N4 substrate. The orthogonal self-assembly process described here provides a promising method by which individual molecules could be spontaneously oriented and connected between closely-spaced, externally-addressable electrodes.