In this work, a self-contained numerical simulation tool for nanoscale Ion-Sensitive Field-Effect Transistor (ISFET) is developed. The tool is based on merging nanoscale ballistic MOSFET analytical equations with the Gouy–Chapman–Stern model equations of ISFET to form a system of nonlinear equations that can be solved iteratively to yield ISFET output current. The numerical solution is accomplished using Newton-Raphson method with efficient trust-region-dogleg algorithm using MATLAB software coding. The tool is used to optimize the sensitivity and linearity of nanoscale ISFETs, and to study their dependence on reference voltage, drain current level, and gate-insulator thickness. Moreover, a comparison between three types of insulators, SiO2, Si3N4, and Al2O3, has been made. The tool is given the name: NIST (Nanoscale ISFET Simulation Tool). It can be used as a guide for design and optimization of nanoscale ISFETs and can be applied for both single-gate and double-gate structures.