Microfluidic paper-based analytical diagnostics have allowed for a diversification of analytical tools by enabling the development of inexpensive and portable devices that build upon existing detection strategies. Taking advantage of existing quantification techniques is an important strategy to ensure that these novel paper-based systems find use and application in the diagnostics world. For more than 50 years high-throughput assays have been developed using polymeric microtiter plates in which signals are quantified using specific absorbance, fluorescence, and luminescence readers. Here, we studied the novel idea of integrating a paper-based analytical assay with a microtiter plate reader. Specifically, electrospun nanofiber mats were designed to match dimensions and criteria of microtiter plate readers. Dye-encapsulating liposomes were used as a model analyte and quantified using absorbance and fluorescence detection strategies. Initially, positively charged poly(vinyl alcohol) (PVA) and polylactic acid (PLA) nanofibers were electrospun and functionalized in specific locations with anti-streptavidin antibodies. Additionally, streptavidin-conjugated liposomes were synthesized to encapsulate sulforhodamine B (SRB) (absorbance wavelength of 488 nm, and a fluorescence excitation and emission wavelengths of 540 nm and 590 nm respectively). Liposomes were then applied and flowed through the nanofiber mats under various conditions to investigate their selective capture, concentration, and detection. Primary investigations demonstrated the ability of PLA as an immobilization matrix to selectively bind streptavidin conjugated liposomes through the use of absorbance measurements. Fluorescence allowed subsequently for accurate readings without the interference of any of the assay materials. The ability to specifically quantify the capture of liposomes using the microtiter plate reader allowed for quantitative optimization of all involved assay steps and buffer systems to increase the reliability of the assay. In the end, the quantification of signals was achieved with a testing volume of 10 µL of SRB encapsulating liposomes, a wash step using 4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES)-sucrose-saline buffer, and 2 µL of detergent for liposome lysis at a concentration of 50 mM. Thus, through the use of streptavidin-conjugated liposomes as a model analyte, it was demonstrated that a PLA nanofiber-based microtiter plate could successfully detect and differentiate between different concentrations of analytes with a detection limit of 0.5 mM and a sensitivity of 4023 Fluorescence units/mM.