The biophysical and pharmacological properties of ion channels and transporters are often studied in exogenous expression systems using either the two-electrode voltage clamp (TEVC) in Xenopus oocytes or the patch clamp techniques. Cells machinery is trusted to produce active proteins that are correctly phosphorylated and glycosylated. However, native physiological cellular processes that might be altered during the course of the experiment are often ignored. Here, we detected and quantified the effects of various electrophysiological recording conditions on the phosphorylation levels of Xenopus oocytes proteins, including membrane proteins, as phosphorylation/dephosphorylation events modulate ion channels gating and cell surface expression. Two strategies were chosen to determine relative protein phosphorylation levels: a direct detection with a phospho-Ser/Thr PKA substrate antibody, and a functional method employing two different leak potassium channels as indicators, chosen based on their opposite responses to protein kinase phosphorylation. We report that holding potential, and bath solution properties such as pH, osmolarity, temperature and ion composition, dramatically affect protein phosphorylation levels in Xenopus oocytes. Our results might explain some of the fluctuations in the biophysical properties of expressed channels, often observed during electrophysiological measurements. Minimizing possible misinterpretations could be achieved using either mutated, kinase insensitive, channels or kinases/phosphatases modulators.