Cavity ring‐down spectroscopy (CRDS) is a highly sensitive laser technique that allows the analysis of isotopic signals and absolute concentration of individual molecular species in small‐volume samples. Here, we describe a protocol to quantify photosynthetic 13C‐uptake rates of marine phytoplankton by using the CRDS technique (13C‐CRDS‐PP). We validated our method by comparing the 13C‐PP rates measured between CRDS and isotope ratio mass spectrometry (IRMS) in samples with different carbon content (30–160 μgC). The comparison revealed that 13C‐CRDS‐PP rates were highly correlated with those obtained by IRMS (Spearman correlation coefficient, ρ = 0.95, p < 0.0001, n = 15), with a mean difference between the two estimates of ± 0.08 mgC m−3 h−1. Moreover, the slope of the relationship between CRDS and IRMS results was not significantly different from 1 (F = 0.03, p = 0.86), and the intercept did not differ from 0 (F = 1.4, p = 0.24), indicating that there was no bias in the CRDS relative to the IRMS‐based measurements. A separate analysis also showed that despite the difference in volume and carbon content between samples (40 ± 10 μgC and 160 ± 40 μgC, respectively), the 13C‐CRDS‐PP technique provides similar results (Mann–Whitney test, U = 30.5, p = 0.90, n = 8). In addition, 13C‐CRDS‐PP rates measured along the Red Sea (∼ 176 mgC m−2 d−1) agreed with 14C‐based PP rates previously reported for similar locations. Thus, this study evidenced that the 13C‐CRDS‐PP method is sensitive enough to quantify carbon fixation rates in oligotrophic regions.