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.