In this work, we describe the fabrication of paper-based aptasensing devices for ampicillin determination that rely on the salt-induced aggregation of gold nanoparticles (AuNPs) in the presence of the target. Circular paper-based devices were created on paper by pen-plotting (using water-repellent ink to create hydrophobic barriers) and were modified with NaCl. The sample was incubated with an ampicillin aptamer and AuNPs and was added to the assay zones of the paper-based devices. In the absence of ampicillin, the aptamer prevented aggregation of the AuNPs and the assay zones remained red. When ampicillin was present, it selectively bound with the aptamer and the AuNPs aggregate producing a purple color. The color of the assay zones was monitored via a smartphone and the color graduation was related to the ampicillin concentration in the sample. Different experimental parameters (type of paper, concentration of reagents, incubation times) were investigated and the analytical features of the method for the determination of ampicillin were established.