The presence and activity of the C4 cycle in C3-C4 intermediate species have proven difficult to analyze, especially when such activity is low. This study proposes a strategy to detect C4 activity and estimate its contribution to overall photosynthesis in intermediate plants, by using tunable diode laser absorption spectroscopy (TDLAS) coupled to gas exchange systems to simultaneously measure the CO2 responses of CO2 assimilation (A) and carbon isotope discrimination (Δ) under low O2 partial pressure. Mathematical models of C3-C4 photosynthesis and Δ are then fitted concurrently to both responses using the same set of constants. This strategy was applied to the intermediate species Flaveria floridana and F. brownii, and to F. pringlei and F. bidentis as C3 and C4 controls, respectively. Our results support the presence of a functional C4 cycle in F. floridana, that can fix 12–21% of carbon. In F. brownii, 75–100% of carbon is fixed via the C4 cycle, and the contribution of mesophyll Rubisco to overall carbon assimilation increases with CO2 partial pressure in both intermediate plants. Combined gas exchange and Δ measurement and modeling is a powerful diagnostic tool for C4 photosynthesis.