Investigating the intracellular contents of single cells is essential for understanding physiologic and pathologic processes at the cellular level. While existing protocols for cell lysis and sample preparation work well for larger samples, scaling to a single-cell level is challenging because of unavoidable analyte dilution and losses. Thus, we are proposing a microfabricated device for the controlled handling and mixing of picoliter cell suspension and lysis solution volumes. Cells and fluids are independently isolated in two microchambers of 25-pL volumes using the geometry of the microchannels and the coordinated action of four on-chip thermopneumatic actuators. Virtual walls formed by liquid-air interfaces in the hydrophobic capillary separate the two volumes, which are subsequently allowed to mix after drawing the air out of the capillary connecting the two microchambers. Following cell lysis, a limited and stable dilution of intracellular components is achieved, simplifying the requirements for subsequent analysis. Two assays at single-cell level, one for direct estimation of the intracellular concentration of a soluble dye and the other for indirect evaluation of intracellular quantities of insoluble actin, demonstrate the use of the microfabricated device for single-cell assays.