Interest in the microfluidic environment, owing to its unique physical properties, is increasing in much innovative chemical, biological, or medicinal research. The possibility of exploiting and using new phenomena makes the microscale a powerful tool to improve currently used macroscopic methods and approaches. Previously, we reported that an increase in the surface area to volume ratio of a measuring cell could provide a wider linear range for fluorescein (Kwapiszewski et al., Anal. Bioanal. Chem. 403:151-155, 2012). Here, we present a broader study in this field to confirm the assumptions we presented before. We studied fluorophores with a large and a small Stokes shift using a standard cuvette and fabricated microfluidic detection cells having different surface area to volume ratios. We analyzed the effect of different configurations of the detection cell on the measured fluorescence signal. We also took into consideration the effect of concentration on the emission spectrum, and the effect of the surface area to volume ratio on the limit of linearity of the response of the selected fluorophores. We observed that downscaling, leading to an increase in the probability of collisions between molecules and cell walls with no energy transfer, results in an increase in the limit of linearity of the calibration curve of fluorophores. The results obtained suggest that microfluidic systems can be an alternative to the currently used approaches for widening the linearity of a calibration curve. Therefore, microsystems can be useful for studies of optically dense samples and samples that should not be diluted.