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This paper proposes a new method: amplitude modulation derivative (AM-derivative) spectrophotometry for UV-Vis spectrophotometer achieved by differentiating the spectral signal modulated to a higher frequency. Both simulating signals and real spectral signals were verified. Values of SNR were obtained (38.87 for AM-derivative spectrophotometry while approximately 0 for conventional derivative spectrophotometry) in simulating experiment. Values of LOD were obtained (0.0129 for AM-derivative spectrophotometry while 0.0134 for conventional derivative spectrophotometry) using paracetamol in real spectral experiment. Results show that AM-derivative spectrophotometry significantly suppresses the noise without weakening the useful signal, therefore supplying a better Signal-to-Noise Ratio and LOD.