Charge carrier concentration in operating organic field-effect transistors (OFETs) reflects the electric potential within the channel, acting as a key quantity to clarify the operation mechanism of the device. Here, we demonstrate a direct determination of charge carrier concentration in the operating devices of pentacene and poly(3-hexylthiophene) (P3HT) by field-induced electron spin resonance (FI-ESR) spectroscopy. This method sensitively detects polarons induced by applying gate voltage, giving a clear FI-ESR signal around g = 2.003 in both devices. Upon applying drain-source voltage, carrier concentration decreases monotonically in the FET linear region, reaching about 70% of the initial value at the pinch-off point, and stayed constant in the saturation region. The observed results are reproduced well from the theoretical potential profile based on the gradual channel model. In particular, the carrier concentration at the pinch-off point is calculated to be β/(β + 1) of the initial value, where β is the power exponent in the gate voltage (V gs ) dependence of the mobility (μ), expressed as μ ∝ V β−2 gs , providing detailed information of charge transport. The present devices show β = 2.6 for the pentacene and β = 2.3 for the P3HT cases, consistent with those determined by transfer characteristics. The gate voltage dependence of the mobility, originating from the charge trapping at the device interface, is confirmed microscopically by the motional narrowing of the FI-ESR spectra.