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We evaluate the robustness of optical density (OD) as a tool in the assessment of the biomass of the cryptophyte Rhodomonas salina under different growth conditions. We measure the OD under three different wavelengths, 550, 665 and 750 nm. We find, as expected, that growth rates of the microalga depend strongly on growth conditions, being highest in saturating light conditions under non‐limiting nitrogen (N) availability. OD – cell count relationships are strong and well defined regardless of the growth conditions and wavelength used for measurement. However, measuring within the absorption range of chlorophyll, at 550 and 665 nm, does give higher regression coefficients under conditions leading to a high cell chlorophyll content, while the coefficients of determinations are slightly higher when cell chlorophyll content is low. We conclude that to use OD as a proxy for biomass under large‐scale production of microalgae, it is important to take into account that the precise relationship between OD and algal biomass does depend on factors such as irradiance and nutrient availability, and hence the physiological state of the microalgae as well as the production conditions. Errors in estimation of biomass may range from 44% to 95%, if these factors are not taken into consideration.