This study describes development and validation of a questionnaire as an adjunct to Traditional Chinese Medicine diagnosis of Yin-Deficiency Syndrome (Yin-DS). The Yin-Deficiency Questionnaire 1 (Yin-DQ1) consists of 10 items. Seventy-nine healthy volunteers and 44 patients diagnosed with Yin-DS were enrolled for the evaluation of discriminant validity and factorial validity. Another group of 83 healthy volunteers participated for test-retest reliability test. Internal consistency was high in both groups (Cronbach's alpha = 0.8615). Test-retest reliability (Spearman's rank correlation coefficient) ranged from 0.54 to 0.79 (p < 0.01). Factor analysis demonstrated that a two factor solution best explained the variance in responses (51.62%). The scores of all items in patients diagnosed with Yin-DS were significantly higher compared with those of healthy volunteers. The data show the internal consistency, test-retest reliability and strong discriminative properties of the Yin-DQ1. Further research determining the optimal cut-off score for Yin-DS and testing its usage as an outcome measure in a clinical trial is needed.