Genetically encoded lipid biosensors are essential cell biological tools. They are the only technique that provide real time, spatially resolved kinetic data for lipid dynamics in living cells. Despite clear strengths, these tools also carry significant drawbacks; most notably, lipid molecules bound to biosensors cannot engage with their effectors, causing inhibition. Here, we show that although PI 3-kinase (PI3K)-mediated activation of Akt is not significantly reduced in a cell population transfected with a PH-Akt1 PIP3/PI(3,4)P2 biosensor, single cells expressing the PH-Akt at visible levels (used for live-cell imaging) have no activated Akt at all. Tagging endogenous AKT1 with neonGreen at its genomic locus reveals its EGF-mediated translocation to the plasma membrane, accumulating at densities of ~0.3 molecules/μm2. Co-transfection with the PH-Akt biosensor or other PIP3 biosensors completely blocks this translocation, despite robust recruitment of the biosensors. A partial inhibition is even observed with PI(3,4)P2-selective biosensor. However, we found that expressing lipid biosensors at low levels, comparable with those of endogenous AKT, produced no such inhibition. Helpfully, these single-molecule biosensors revealed improved dynamic range and kinetic fidelity compared with over-expressed biosensor. This approach represents a less invasive way to probe spatiotemporal dynamics of the PI3K pathway in living cells.