310 test, published by Organisation for Economic Co-operation and Development, is the ready biodegradability test with the most extensive practicability. However, the test guideline does not provide a setting method of test system’s headspace volume. Based on the test validity requirements for dissolved oxygen concentration and distribution of test item in air/water two-phase system, the calculation formulas for test system’s headspace volume were obtained. The calculation principles were Henry’s law and ideal gas equation, and the formulas contained four parameters: oxygen consumption of microorganisms, test item concentration, test item’s theoretical oxygen demand and test item’s Henry’s law constant. 2-methylnaphthanlene and limonene were selected as examples for explanation of formula use. The result can provide help for test design and method standardization.