Simple and reproducible tools to assess antiretroviral adherence are needed. A level of tenofovir diphosphate (TFV-DP) in dried blood spots (DBS) <1,250 fmol/punch is predicted to identify imperfect adherence. Herein we evaluated TFV-DP in DBS as a measure of adherence among HIV-infected women. DBS and peripheral blood mononuclear cells (PBMCs) were collected twice (∼1 week apart) in 35 well-controlled HIV-infected women [median age 42 years, 14 African American/black (AA)] receiving daily coformulated tenofovir/emtricitabine and either atazanavir/ritonavir (n=20) or raltegravir (n=16). TFV-DP in DBS and PBMCs was quantified by LC-MS/MS. Six-month adherence was measured as average days between monthly pharmacy refills. Data were loge transformed for analysis and presented as median (range); the correlation between continuous variables was analyzed using the Pearson correlation coefficient. The average TFV-DP between the two visits (aTFV-DP) in DBS and PBMCs was 1,874 (706-3,776) fmol/punch and 125 (1-278) fmol/10(6) cells, respectively. AA women had lower levels of aTFV-DP in DBS compared to whites (1,660 vs. 1,970 fmol/punch; p=0.04), with a viremic patient having the lowest drug levels (706 fmol/punch). Days between pharmacy refills were 34 (30-54) vs. 30 (26-40) in women with TFV-DP in DBS <1,250 vs. ≥1,250 fmol/punch (p=0.006). TFV-DP in DBS was negatively correlated with an increasing number of days between refills (r=-0.56, p=0.002). TFV-DP DBS was a reliable and objective measure of adherence in HIV-infected women based on a strong inverse relationship with pharmacy refill adherence.