Abstract Background Rapid, CLIA-waived quantitative testing can help eliminate delays in receiving care, improve telemedicine, enhance the management of chronic diseases and clinical trials, and reduce healthcare costs. Intelligent Optical Systems has developed an innovative Enhanced Lateral Flow Assay (ELFA) platform and partnered with the University of California, Irvine Nephrology Division and Wake Forest Institute for Regenerative Medicine for the clinical validation of the assay. The ELFA platform quantitatively measures the immunosuppressive drug tacrolimus (TAC), and health biomarkers such as proinflammatory cytokines IL6, IL8, matrix metalloproteinase-3 (MMP3), glial fibrillary acidic protein (GFAP, an indicator of traumatic brain injury), and cystatin C (CysC, an indicator of kidney function) from fingerstick blood at the point of care. The testing panel is continuously expanding. Methods The ELFA platform is an in vitro, quantitative, single plex or multiplexed lateral flow immunoassay that consists of three main components: (i) simple fingerstick whole blood sampling kit; (ii) lateral flow test strip with running buffer and dried fluorescent labeled affinity reagents; and (iii) miniaturized readout device that can be further developed with a controlled user interface, enabling untrained users to easily record results and instantly transmit them to the physician. Depending on the analytes, the ELFA point-of-care platform has undergone bench testing or clinical validation studies, including: (1) Single plex for TAC: Clinical validation using 5 uL of capillary fingerstick whole blood from kidney transplant recipients (KTR, n = 50) compared to standard LC-MS/MS laboratory method. (2) Multiplexed ELFA for TAC, IL6, and MMP3: In vitro assessment of limit of detection (LoD), detection range, specificity, and repeatability (n = 5) with 10 uL spiked whole blood. (3) Single plex ELFA for GFAP, IL8, and CysC: In vitro determination of the LoD, detection range, CV%, and correlation of spiked and estimated analyte levels with 10 uL of spiked whole blood. For all analytes, a 4-parameter logistic calibration curve with n = 3 for each level of analyte was constructed to determine the LoD, detection range, and CV%. Pearson correlation and Bland-Altman analysis were applied to correlate and compare data between spiked and estimated levels, or between ELFA and standard lab test methods. Results Study (1) demonstrated excellent correlation between ELFA and LC-MS/MS, with a correlation coefficient r-value of 0.89, and at 95% limit of agreement, a mean difference of 0.7 ng/mL in the clinically compared samples from 50 KTRs. Study (2) showed an LoD and range of detection of 2 ng/mL to 20 ng/mL, 1 ng/mL to 5 ng/mL, and 10 ng/mL to 200 ng/mL for the multiplexed detection of TAC, IL6, and MMP3, respectively. The repeatability assessment showed a <10%CV for all analytes. Study (3) showed an LoD and range of detection of 1 ng/mL to 10 ng/mL, 0.5 ng/mL to 5 ng/mL, and 0 to 5 mg/L for GFAP, IL8, and CysC, respectively. Conclusion The ELFA platform delivers minimally invasive, convenient sampling in a POC device using capillary fingerstick blood, and provides accurate, rapid measurements of TAC, which demonstrate good repeatability and strong correlation against LC-MS standard reference measurements.