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Alkaline phosphatase (ALP) is a reporter enzyme widely used in point-of-care applications. Real-time monitoring of ALP accelerates understanding of emerging diseases and of cancer progression. This can be achieved through biosensor-based strategies. This paper outlines an approach that facilitates the real-time monitoring of ALP release from the differentiation of cultured human colon cancer cells (Ht-29). Electric cell-substrate impedance sensing was used to generate changes in Ht-29 cells during ALP release and the data were compared using amperometry. The findings indicate a correlation between cell response and shift in impedance magnitude and current. Relevant results had similar attitude during cell monitoring, which influenced cell morphology and cell viability. Our analysis is a clear advance on the current methods such as enzyme-linked immunosorbent assay. The developed method can contribute to the development of microfluidic testing that would facilitate work on small-scale techniques. [ABSTRACT FROM AUTHOR]