The control of free ionized intracellular calcium concentration ([Ca2+ ]i ) is an established mechanism of cellular activation, regulating a diverse range of cellular events. Consequentially, experimental measurement of [Ca2+ ]i is a potent technique for the medical science laboratory. The NOVOstar microplate reader is a versatile system, which may be easily configured to measure [Ca2+ ]i . Moreover, the relatively low cost of this system makes it an attractive one for researchers adhering to a modest budget, whilst allowing medium throughput to be achieved. These methods serve as a starting point for researchers wishing to measure intracellular calcium concentration in adherent cell-lines using the NOVOstar plate reader. Briefly, adherent cells are seeded into well plates 1 day prior to calcium determinations being made. On the day of the experiment, autofluorescence values of individual wells of cells are determined prior to the cells being loaded with the fluorophore, fura-2. [Ca2+ ]i determinations are acquired by activating a predefined program within the NOVOstar software; full parameters are provided within this chapter for this purpose. Fluorescence ratio values may be easily calibrated to give absolute intracellular calcium concentrations (nM). Calibration involves determining experimental fluorescence at calcium-saturating and calcium-free conditions; ionomycin and EGTA are used to produce these two conditions respectively. Finally, mathematical calculation of absolute intracellular calcium concentration is described by use of the Grynkiewicz equation.