Background: Organic solvents used for extraction of tacrolimus from whole blood samples lower the apparent affinity of the antibody used in a diagnostic immunoassay, thereby affecting the detection limit. Methods: We used in vitro recombinant antibody engineering to screen and isolate clones from diverse libraries with mutagenic complementarity regions (CDRs) from tacrolimus 1-60-46 hybridoma cell line, with improved binding to tacrolimus in the presence of 10% methanol organic solvent solution. Results: We isolated a number of clones with mutations in variable heavy (VH) CDR 2, variable light (VL) CDR 1, and VL CDR 3 with improved binding. Various combinatorial pairings constructed from these individual mutations contained >10-fold improvements in both the dissociation rate and overall equilibrium affinity constants. Selected clones produced as IgG have increased functional sensitivity, with a 3- to 6-fold reduction in the limit of detection relative to the parental tacrolimus 1-60-46 monoclonal antibody in the Architect® Tacrolimus immunodiagnostic assay. Conclusions: The recent advent of recombinant in vitro antibody display technologies in general, and yeast surface display in particular, allows the flexibility to engineer new or augment specific analytical characteristics, such as affinity, specificity, or stability, into previously isolated and otherwise desirable antibodies to enhance assay performance. These in vitro selections can also be performed under conditions meant to mimic the assay in which the reagent will ultimately be used, to increase the likelihood of successful assay development.