Microgreens are quickly-grown greens that are suitable for urban areas. The accumulation of chlorophyll in plants is essential for both nutritional compounds and the microgreen’s attractive appearance. A plant factory is a controlled-environment cultivation method that employs LEDs to enhance photosynthesis, vegetative development, seed germination, and chlorophyll accumulation. This work used VIS-NIR for the quantitative determination of chlorophyll content in green and red spinach microgreens grown under four different LED light intensities with four different ratios, with 100% blue, red, and white LED, and 67%:20%:13% of red:blue: white LED, as artificial grow for 12 hours illumination. The performance of MSC, AN, SNV, smoothing, and the first and second Savitzky-Golay’s derivatives (SGD) were examined. PLSR explained accurately predicted R2C, R2P, RMSEC, and RMSEP up to 0.957, 0.892, 1.054, and 1.661 for chlorophyll a using SGD 1st; 0.813, 0.652, 0.979, and 1.338 for chlorophyll b using SNV; and 0.931, 0.876, 1.155, and 1.550 for total chlorophyll using SNV. The wavelength region of 400 to 700 nm was dominant based on the best regression coefficient (β) for predicting green and red spinach microgreens. Results demonstrated that VIS-NIR spectroscopy, combined with chemometric techniques, PLSR, can be used to evaluate the chlorophyll content of green and red spinach microgreens.