<p>In social insects, colonies are composed of numerous individuals that cooperate to form a unified entity with coordinated control mechanisms. Because colony-level behaviors emerge from the accumulation of interactions among individuals, colony size can strongly influence traits such as resource allocation and caste composition. Therefore, to understand the regulatory mechanisms and investment strategies of colonies, it is essential to monitor their dynamics with explicit consideration of colony size. However, in subterranean termites, colony size estimation remains challenging due to their cryptic nesting habits. Even under laboratory rearing conditions, assessing colony size often damages individuals or nest structures, making repeated longitudinal monitoring difficult. Here, we developed a non-invasive method for estimating colony biomass in laboratory-reared colonies of <i>Reticulitermes speratus</i> based on CO₂ emissions. By recording CO₂ concentrations for 90&#xa0;min in a sealed container, we found a strong correlation between total termite biomass and CO₂ output (r = 0.982, R² = 0.965), whereas correlations with nest volume proxies were weaker. Repeated measurements every two days over four sessions revealed no significant decline in estimated biomass, and a non-inferiority test confirmed that CO₂ assays did not reduce colony biomass. This approach provides a practical solution for repeated, non-invasive estimation of termite colony biomass, enabling longitudinal studies of caste differentiation, reproductive transitions, and behavioral regulation in relation to colony size, as reflected by colony biomass. Because CO₂ emissions from respiration are a universal biological trait, the method may also be applicable to other termite species and more distantly related taxa.</p>