Résumé : Some microalgae store large amounts of neutral lipids inside lipid droplets. Since these lipids can be used to produce biodiesel in a sustainable way, research is developing on fast non-destructive methods to quantify and monitor the amount of lipids in microalgal cultures. In this paper, we have developed with digital holographic microscopy a fast quantitative method to assess the evolution of the lipid content inside the diatom Phaeodactylum tricornutum living cells. The method uses a specific processing of recorded hologram sequences based on the refocusing capability of digital holographic microscopy. Each lipid droplet volume is evaluated inside the cells on representative samples of the culture. We have validated the method thanks to correlative quantitative phase contrast-fluorescence imaging and extrapolated it to larger calibrated spherical refractive particles, to demonstrate the flexibility of the method.