Prenatal androgens influence the second to fourth digit ratio (2D:4D) of hands with men having lower ratios than women. Numerous methods are used to assess 2D:4D including, physical measurements with calipers, and measurements made from photocopies, scanned images, digital photographs, radiographs, and scaled tubes. Although each method appears relatively reliable, agreement upon a gold standard is necessary to better explore the putative effects of prenatal androgens. Our objective was to assess the level of intra and interobserver reliability when evaluating 2D:4D using four techniques: (1) physical measurements, (2) photocopies, (3) printed scanned images, and (4) computer-assisted image analysis. Physical measurements, photocopies, and printed scanned images were measured with Vernier calipers. Scanned images were also measured with computer-based calipers. Measurements were made in 30 men and 30 women at two different time points, by three experienced observers. Intraclass correlation coefficients were used to assess the level of reliability. Intraobserver reliability was best for computer-assisted (0.957), followed by photocopies (0.939), physical measurements (0.925), and printed scans (0.842; P = 0.015). Interobserver reliability was also greatest for computer-assisted (0.892), followed by photocopies (0.858), physical measurements (0.795), and printed scans (0.761; P = 0.001). Mean 2D:4D from physical measurements were higher than all other techniques (P < 0.0001). Digit ratios determined from computer-assisted, physical measurements, and printed scans were more reliable in men than women (P = 0.009, P = 0.017, and P = 0.012, respectively).In summary, 2D:4D determined from computer-assisted analysis yielded the most accurate and consistent measurements among observers. Investigations of 2D:4D should use computer-assisted measurements over alternate methods whenever possible.Prenatal androgen exposure influences development of the fingers and leads to distinct differences in hand patterns among men and women. In general, the ratio between the second (2D = index finger) and fourth (4D = ring finger) digits in men is lower than that observed in women (Lutchmaya et al., 2004;Manning et al., 1998). Anatomical evidence of prenatal androgen exposure has gained significance in recent times because there is growing evidence in animal models that adult traits and behaviors including, disease susceptibility may be programmed during fetal development (Abbott et al., 2006;Manikkam et al., 2004 Manning and Bundred, 2000;Robinson, 2006). Previous studies in humans have shown low digit ratios (2D:4D) to be associated with a variety of traits including, enhanced visual spatial ability (Burton et al., 2005;van Anders and Hampson, 2005), increased athleticism (Honekopp et al., 2006a), female homosexuality (Brown et al., 2002;Hall and Love, 2003;Kraemer et al., 2006;Williams et al., 2000), decreased susceptibility to coronary artery disease (Fink et al., 2003;Manning and Bundred, 2000), and greater fertility potent...