This study represents a pioneering initiative in the printing industry, especially in Poland, which assessed the environmental impacts and eco-efficiency of proof printing through the life cycle assessment (LCA) methodology. The process of proof printing on a target substrate was compared with the traditional hard proofing process, which requires trial printing in production conditions. The analysis adhered to the ISO 14040 and 14044 standards, assessing greenhouse gas (GHG) emissions, raw material use (e.g., plastics, water), and environmental toxicity. The innovative proofing on the target substrate process exhibits a lower environmental impact, as confirmed by the GHG emissions and plastic and water demand of the process. The GHG emissions were reduced from 2610 kg of CO2e to 68.4 kg of CO2e per functional unit (FU). The water demand for the proofing on the target substrate process was 40 times lower, and the plastic demand was also 40 times lower, decreasing to 20 kg per FU. The toxicity impact of the method based on the proofing system on the target substrate on living organisms is more than six times lower than that of the conventional method. The proof printing on the target substrate process offers an environmentally friendly alternative to the traditional hard proofing process, with lower GHG emissions and a lower environmental impact.