Advanced precision agriculture requires the objective measurement of the structural and functional properties of plants. Biochemical profiles in leaves can differ depending on plant growing conditions. By quantitatively detecting these changes, farm production processes can be optimized to achieve high-yield, high-quality, and nutrient dense agricultural products. To enable the rapid and non-destructive detection on site, this study demonstrates the development of a new custom-designed portable handheld Vis–NIR spectrometer that collects leaf reflectance spectra, wirelessly transfers the spectral data through Bluetooth, and provides both raw spectral data and processed information. The spectrometer has two preprogramed methods: anthocyanin and chlorophyll quantification. Anthocyanin content of red and green lettuce estimated with the new spectrometer showed an excellent correlation coefficient of 0.84 with those determined by a destructive gold standard biochemical method. The differences in chlorophyll content were measured using leaf senescence as a case study. Chlorophyll Index calculated with the handheld spectrometer gradually decreased with leaf age as chlorophyll degrades during the process of senescence. The estimated chlorophyll values were highly correlated with those obtained from a commercial fluorescence-based chlorophyll meter with a correlation coefficient of 0.77. The developed portable handheld Vis–NIR spectrometer could be a simple, cost-effective, and easy to operate tool that can be used to non-invasively monitor plant pigment and nutrient content efficiently.