Purpose <p>Small molecule-based radiotracers offer several potential advantages in positron emission tomography (PET) imaging, and are therefore a promising approach for non-invasively and accurately monitoring of programmed death ligand 1 (PD-L1) expression in vivo. In this study, two small-molecule radiotracers were developed to assess PD-L1 expression and dynamics during treatments.</p> Methods <p>[¹⁸F]LG-2 and [¹⁸F]LG-3 were designed based on a phenoxymethyl-biphenyl scaffold with a tris-(hydroxymethyl)-aminomethane terminal group. The radiolabeling was achieved by a two-step method through the “click” chemistry. Cellular uptake assays in different tumor cells were performed to determine the specificity of the two tracers to PD-L1. The ability of [¹⁸F]LG-2 and [¹⁸F]LG-3 to detect PD-L1 expression in vivo as well as to monitor PD-L1 dynamics during chemotherapy and immunotherapy was investigated <i>via</i> PET imaging.</p> Results <p>The radiolabeling of [¹⁸F]LG-2 and [¹⁸F]LG-3 was achieved with overall radiochemical yield of 15 ± 3% for [¹⁸F]LG-2 and 18 ± 5% for [¹⁸F]LG-3. In vitro cell uptake studies in tumor cells with varying PD-L1 levels demonstrated the specific binding of these tracers to PD-L1. PET imaging in mice bearing B16-F10 tumors displayed comparable tumor uptake of 6.45 ± 0.38%ID/mL for [¹⁸F]LG-2 and 5.64 ± 0.02%ID/mL for [¹⁸F]LG-3, while [¹⁸F]LG-3 showed nearly a 50% reduction in uptake in the liver and intestines compared to [¹⁸F]LG-2. PET signals of [¹⁸F]LG-3 in A375-hPD-L1, A375-hPD-L1/A375 and A375 tumor-bearing mice demonstrated a strong and linear correlation with PD-L1 expression levels. The dynamic of PD-L1 status in tumors after cisplatin and PD-L1 inhibitor treatments were accurately evaluated with [¹⁸F]LG-3 PET imaging.</p> Conclusion <p>The small-molecule radiotracer [¹⁸F]LG-3 is a promising candidate for evaluating PD-L1 expression and monitoring the dynamic of PD-L1 status during the treatment process.</p>