Abstract Insulin resistance has been associated with higher plasma amino acid ( AA ) concentrations, but majority of studies have used indirect measures of insulin resistance. Our main objective was to define the relationship between plasma AA concentrations and a direct measure of insulin resistance in women and men. This was a cross‐sectional study of 182 nondiabetic individuals (118 women and 64 men) who had measurement of 24 AA s and steady‐state plasma glucose ( SSPG ) concentration (insulin resistance) using the insulin suppression test. Fourteen out of 24 AA concentrations were significantly ( P < 0.05) higher in men than women; only glycine was lower in men. Majority of these AA s were positively associated with SSPG ; only glycine concentration was negatively associated. Glutamic acid, isoleucine, leucine, and tyrosine concentrations had the strongest correlation with SSPG ( r ≥ 0.4, P < 0.001). The degree of association was similar in women and men, independent of obesity, and similar to traditional markers of insulin resistance (e.g., glucose, triglyceride, high‐density lipoprotein cholesterol). Compared with women, men tended to have a more unfavorable AA profile with higher concentration of AA s associated with insulin resistance and less glycine. However, the strength of association between a direct measurement of insulin resistance and AA concentrations were similar between sexes and equivalent to several traditional markers of insulin resistance.