Purpose: The purpose of this study is to identify the effect of Progressive Space Organization (ante-rooms) in Negative Pressured Isolation Unit(NPIU) such as National and Regional Isolation Units in Korea in order to build basic data for the evidence based design of Airborne Infection Isolation Units which should prepare and respond effectively to the public health crisis due to the hazardous airborne infectious disease. Methods: 1) Gas(SF6) test and analysis on the 23 Korean Isolation Units under operation. 2) Assessment of the isolation level of the space components by checking the Gas concentration. 3) Analysis of the Isolation Effectiveness according to Space Organizational levels. Results: 1) The higher segregation level is, the lower Gas(SF6) concentration is. 2) Too many segregations(anterooms) of Isolation Unit are not efficient for the prevention of infectious bacillus spread. For example, 4 level of segregation has similar segregation effect to the 3 level of segregation. Implications: Many anterooms in front of the isolated patient bedroom will guarantee the safe environment against the danger of hazardous airborne nosocomial infection. On the other hand, too many segregations is inefficient, expensive, inconvenient, narrow(unflexible) and so on. This study can be used as basic data for further development of design guidelines of isolation units