Our objective was to develop and validate a disposable fluorescence-based test strip coupled with a portable imaging device for quantification of plasma progesterone (P4). First, we developed and optimized a competitive lateral flow immunoassay (LFIA) test strip to measure P4 in bovine plasma. The LFIA design included a sample pad, a conjugate pad that dry-stores Rphycoerythriin-anti-P4 conjugates, a glass-fiber spacer pad, a nitrocellulose membrane with printed test and control lines, and a cellulose-fiber absorbent pad. To perform a test, 20 µL of plasma and 50 µL of running buffer (RB) were added on the sample pad, and the test strip was left in a light-free environment. After 3 min, 45 µL of RB were added to initiate sample flow. After allowing 15 min to stabilize the colorimetric signal, strips were inserted into an LFIA reader to determine P4 concentration based on test-to-control-line signal (T/C ratio). The reader was linked to a laptop to interpret and display results. In a series of experiments (n = 6), the ability of the LFIA to predict the presence of a functional (P4 ≥ 1 ng/mL) corpus luteum (CL) in bovine plasma samples was evaluated. For each experiment, a calibration curve was constructed using plasma with known concentrations of P4 (0.1–3.7 ng/mL; n = 5). There was a linear relationship between average T/C ratio and P4 levels (average R² = 0.82; range 0.44–0.99). The T/C ratio decreased as P4 concentrations increased. Next, plasma samples from lactating dairy cattle (n = 58) were tested in triplicate to predict the presence or absence of a functional CL using a radioimmunoassay for P4 as reference test. Overall, the LFIA assay correctly classified 90% (P < 0.01; 95%CI 79–96) of the samples, with 93% sensitivity (P < 0.01; 95%CI 77–99), 86% specificity (P < 0.01; 95%CI 68–96), 87% positive predictive value (P < 0.01; 95%CI 70–96) and 93% negative predictive value (P < 0.01; 95%CI 76 to 99). The agreement between the LFIA and the reference test was substantial (kappa = 0.79; 95%CI 0.64–0.95; P < 0.01).The intra-assay CV averaged 16.0% (range 0.5–42.8%). These data suggest that the current LFIA system can accurately predict the presence of a CL based on circulating concentrations of P4. Supported by USDA-NIFA Project 2016–08814.