Nutrients derived from fish feed are insufficient for optimal plant growth in aquaponics; therefore, they need to be supplemented. Thus, estimating the amount of supplementation needed can be achieved by looking at the nutrient contents of the plant. This study aims to develop trustworthy machine learning models to estimate the nitrogen (N), phosphorus (P), and potassium (K) contents of aquaponically grown lettuce. A FieldSpec4, Pro FR portable spectroradiometer (ASD Inc., Analytical Spectral Devices Boulder, Boulder, CO, USA) was used to measure leaf reflectance spectra, and 128 lettuce seedlings given four NPK treatments were used for spectra acquisition and total NPK estimation. Principal component analysis (PCA), genetic algorithms (GA), and sequential forward selection (SFS) were applied to select the optimal wavebands. Partial least squares regression (PLSR), back-propagation neural network (BPNN), and random forest (RF) approaches were used to develop the predictive models of NPK contents using the selected optimal wavelengths. Good and significantly correlated predictive accuracy was obtained in comparison with the laboratory-measured freshly cut lettuce leaves with R2 ≥ 0.94. The proposed approach provides a pathway toward automatic nutrient estimation of aquaponically grown lettuce. Consequently, aquaponics will become more intelligent, and will be adopted as a precision agriculture technology.