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A spectrophotometric method for the determination of glyphosate based on the monitoring of a complex formation between bis 5-phenyldipyrrinate of nickel (II) and the herbicide was developed. The method showed a short response time (10 s), high selectivity (very low interference from other pesticides and salts), and high sensitivity (LOD 2.07 × 10^−7 mol/L, LOQ 9.87 × 10^−7 mol/L, and a K_d from 1.75 × 10^−6 to 6.95 × 10^−6 mol/L). The Job plot showed that complex formation occurs with a 1:1 stoichiometry. The method was successfully applied in potable, urban, groundwater, and residual-treated water samples, showing high precision (0.34–2.9%) and accuracy (87.20–119.04%). The structure of the complex was elucidated through theoretical studies demonstrating that the nickel in the bis 5-phenyldipyrrinate forms a distorted octahedral molecular geometry by expanding its coordination number through one bond with the nitrogen and another with the oxygen of the glyphosate’ carboxyl group, at distances between 1.89–2.08 Å.