The LAI‐2000 Plant Canopy Analyzer provides a rapid estimate of leaf area index (LAI), but its accuracy and utility for estimating soybean [Glycine max (L.) Merr.] defoliation is unknown. This study evaluated minimum plot‐size requirements for manually defoliated soybean experiments using the LAI‐2000; compared LAI estimates using the LAI‐2000 with directly determined values for 0, 33, 67, and 100% manual defoliation levels; and compared estimates between two LAI‐2000 detector types having spectral sensitivity differences. The minimum plot size for obtaining accurate indirect estimates of LAI in defoliated canopies (0.89‐m height) with 0.38‐m row centers is six rows by 2.28 m and with 0.91‐m row centers and a canopy height of 0.81 m is four rows by 2 m plus an additional 1‐m defoliated area at the ends of the two middle rows. Indirect measurements of LAI with the wide‐blue detector, which is included on newer LAI‐2000 units, were statistically greater than directly determined values in 10 of 12 comparisons. Measurements with the narrow‐blue detector were the same as directly determined values in three of five comparisons in 1998 and 1999 but were statistically greater in 11 of 12 comparisons in 2000. Estimates with the wide‐blue detector were 9 and 2% greater than those with the narrow‐blue detector in 1999 and 2000, respectively.