Nonesterified fatty acids (NEFA) are measured routinely in the study of nutrition and growth physiology. To improve the efficiency with which this variable is measured, a two-reaction, enzymatic-based assay was adapted and validated to quantify NEFA in bovine blood plasma using 96-well microtiter plates. The effects of incubation time, temperature, and of sample volume were examined in addition to possible interfering substances, recovery, reagent stability, and range of linearity. Incubation for 30 min at 21 degrees C for each of the two reactions resulted in maximal slope and r2 values (1.94 and .999, respectively). Percentage of recovery was 107% when using 5 microL and 100% when using 10 microL of heparinized bovine plasma in the assay. Uniform linear standard curves (r2 > .99) were obtained using reagents stored at 4 degrees C for 9 d. An equal volume of 1 mM acetate, propionate, valerate, and butyrate added to plasma did not affect results. The assay was linear from 125 to > 1,000 microEq/L. Intra- and interassay CV for the 5-microL sample volume were 3.6 and 3.7%, respectively. This modified assay provides results comparable to the standard assay yet reduces reagent and labor requirements and increases sample capacity.