Anthropogenic CO2 emissions are the main contributors to climate change. Among the various efforts to reduce atmospheric CO2 levels, cultivation of microalgae is the most promising approach owing to its high photosynthetic rates and CO2 fixation efficiencies than terrestrial counterparts. However, the accurate quantification method of CO2 fixation during the cultivation of microalgae in photobioreactors (PBRs) is lacking. Present methods for the determination of CO2 fixation during microalgae cultivation include direct and indirect methods, where 79% of direct method studies of the bibliometric analysis compared to 21% of indirect method studies. Direct methods evaluate the carbon content in microalgae biomass using assumptive values, though it results in significant errors as high as 50% in quantifying the CO2 fixation. This can be improved by measuring the carbon contents using elemental and total organic carbon analysis. On the other hand, indirect methods quantify CO2 concentration at inlet and outlet of PBRs by using gas chromatography or infrared sensors. It is rather difficult to validate the accuracy of direct and indirect methods due to the lack of comparative works and analysis among the methods. Additionally, there are no current studies that provide in-depth discussion and perspectives on the CO2 fixation methods. Therefore, the main aim of this critical review is to analyse, contrast and discuss the differences as well as inaccuracies of direct and indirect microalgae CO2 fixation quantifications in PBRs. This is followed by the recommendations for further improvements, and standard guidance for future studies in applying appropriate CO2 fixation quantification methods. Display omitted •A comprehensive guide for CO2 fixation in microalgae photobioreactors is discussed.•Microalgae CO2 fixation can be quantified by direct and indirect methods.•Direct methods quantify CO2 fixation via microalgae biomass carbon content.•Indirect methods quantify CO2 fixation by photobioreactor CO2 gas concentrations.•Identified the gaps and suggested methods for accurate CO2 fixation quantifications.