Analysis of intracellular materials at the single-cell level presents opportunities for probing the heterogeneity of a cell population. Lysis by electroporation has been gaining popularity as a rapid method for disruption of the cell membrane and release of intracellular contents. In this report, we selectively released specific intracellular molecules for interrogation at the single-cell level by tuning the parameters of electroporation. We examined the release of a small molecule, calcein (MW similar to 600), and a 72-kDa protein kinase, Syk, tagged by enhanced green fluorescent protein (EGFP) from chicken B cells during electroporation at the single-cell level. We studied the effects of the field intensity and the field duration on the release of the two molecules. We found that calcein in general was released at lower field intensities and shorter durations than did SykEGFP. By tuning the electrical parameters, we were able to deplete calcein from the cells before SykEGFP started to release. This approach potentially provides a high-throughput alternative for probing different intracellular molecules at the single-cell level compared to chemical cytometry by eliminating complete disruption of the cell membrane.