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To develop rapid, disposable sensors for biomarkers assessment in biological fluids of non-invasive collection, natural waters and food samples was the driving force of the project PHsense – Design and development of disposable, user-friendly microfluidic sensors to monitor Public Health indicators in a One Health approach. The idea was to produce fast-response and accurate sensor devices to enable reliable and real-time response, using self-readable paper-based microfluidic devices (µPADs) applied to both environmental and biological samples, which is a breakthrough for understanding public health potential issues, thus enabling an efficient course of action. One of the main goals of the World Health Organization (WHO) is that “all people have the right to have access to an adequate supply of safe drinking water”. Due to the economic situation in Portugal and the current water tariff scenario, recent studies have shown that the consumption of groundwater has been increasing by the population with the use of water from wells for domestic use. The homeowners are responsible for testing the water quality to ensure that it is safe for consumption and food preparation. This water can be contaminated by a variety of toxic substances and early detection is crucial to prevent harm to public health. The combined monitoring of biological fluids will enable to establish a potential contamination, hazard bioaccumulation and identify the contamination source. In this context, µPADs can be the ideal approach, exploiting advantage of being used by untrained personnel, being almost equipment-free with low reagents and sample consumption. The developed devices represent a vital strategy for attaining effective monitoring and frequent analysis, enabling fast chemical/biochemical response resulting in a faster analytical decision. These new tools must be accurate and aligned with the ASSURED criteria established by WHO, namely “affordable, sensitive, specific, user-friendly, rapid and robust, equipment-free, and derivable to end-users”.