The atmosphere at Caltech changed during World War II. Fewer young men attended Caltech during the war years and many members of Caltech's staff focused their attention on projects that aided the war effort. Some chose to leave Caltech, however Pauling remained in Pasadena while conducting many research projects for the United States government. He aided the war effort by continuing to work on immunology and by branching out into new areas that built upon his previous endeavors. Two of his projects involved hemoglobin.

Pauling tried to find a serum that could substitute for blood and be used for blood transfusions. Working for the Office of Scientific Research and Development (OSRD), Pauling and Campbell, one of his collaborators on immunological research, developed a successful serum substitute called oxypolygelatin. Oxypolygelatin was not used, however, because by 1943 there were enough blood donors to make using the serum unnecessary. Even though the government lost interest in oxypolygelatin, Pauling did not. During the 1940s, he, Campbell, and others continued to work on the blood substitute and improved the gelatin. In December 1946 Pauling, Campbell, and another colleague submitted a patent application for Oxypolygelatin.

Pauling also developed a spectrophotometric device that determined the amount of carbon monoxide in the air based upon the concentration of carbon monoxide in a sample of blood. He made this instrument for airplanes and tanks on the request of the National Defense Research Committee. Spectrophotometry, the technique used for the apparatus, yields concentration information through absorption spectra. In the end Pauling found that the device was unsuitable for use because of its bulk, sensitivity and the instability of the reagent, oxyhemoglobin.

The United States government acknowledged Pauling for his scientific work that aided the war. The most prestigious award he received was the Medal for Merit from President Harry S. Truman, who called Pauling's work "brilliant."