We developed microfluidic paper-based analytical devices (μPADs) for the chelate titrations of Ca2+ and Mg2+ in natural water. The μPAD consisted of ten reaction zones and ten detection zones connected through narrow channels to a sample zone located at the center. Buffer solutions with a pH of 10 or 13 were applied to all surfaces of the channels and zones. Different amounts of ethylenediaminetetraacetic acid (EDTA) were added to the reaction zones and a consistent amount of a metal indicator (Eriochrome Black T or Calcon) was added to the detection zones. The total concentrations of Ca2+ and Mg2+ (total hardness) in the water were measured using a μPAD containing a buffer solution with a pH of 10, whereas only Ca2+ was titrated using a μPAD prepared with a potassium hydroxide solution with a pH of 13. The μPADs permitted the determination of Ca2+ and Mg2+ in mineral water, river water, and seawater samples within only a few minutes using only the naked eye—no need of instruments.