Objectives: To validate the ecological modification of the Papanicolaou stain (Eco-Pap) for the diagnosis of cervical cancer. Study Design: A prospective study was performed at the Mother and Child "San Bartolomé" Hospital, Lima, Peru. Reagent handling strategies were divided into three phases: we used (1) Harris progressive hematoxylin (for nuclear staining), (2) a polychromatic solution (a mix of EA-36/Orange G-6 to suppress the use of several alcohol baths), and (3) direct mounting (with Entellan® solution). The cellular details were analyzed by the staining quality index, an external quality control, and the Bethesda System 2014. Results: We evaluated 72,901 cervical smears stained with the Eco-Pap. The validation of the Eco-Pap against the conventional Pap stain was optimal (κ = 0.89; 95% CI: 0.87-0.92), showing a sensibility and specificity of 88.3% (95% CI: 85.1-90.0) and 98.7% (95% CI: 98-99.2), respectively. The Eco-Pap dramatically reduced the environmental pollution caused by 72 L of xylene, hydrochloric acid, and ammonia (6 L each) and mercury oxide. Conclusion: The Eco-Pap is an innovative and efficient staining method reducing the use of toxic reagents with carcinogenic potential during cervical cancer screening by exfoliative cytology.