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The main objective of the present study is to quantify doxycycline (DOX) release from β-tricalcium phosphate (β-TCP) after EDTA root surface treatment.Thirty systemically healthy patients with ≥1 paired contralateral interproximal intrabony defect ≥4 mm deep along with an interproximal probing depth ≥6 mm and clinical attachment level ≥4 mm were randomized into two groups. Group 1 (G1) consisted of sites treated with open flap debridement followed by placement of DOX blended with β-TCP (DOX-β-TCP), whereas group 2 (G2) sites were treated with flap surgery followed by the placement of DOX blended with β-TCP after EDTA etching of the exposed root surfaces (DOX-β-TCP + EDTA). Samples of gingival crevicular fluid (GCF) were obtained 1, 3, 7, 14, and 21 days after surgery. Quantitative measurements of DOX were taken with high-performance liquid chromatography. Clinical evaluation and follow-up for 6 months were performed.At 21 days, the DOX-β-TCP + EDTA-treated group showed a 194.7 μg/mL value. The DOX-β-TCP + EDTA-treated group retained more DOX during the periods of 3, 7, 10, 14, and 21 days than the DOX-β-TCP-treated group. Six months after therapy, DOX-β-TCP + EDTA-treated sites showed more significant clinical improvements compared to DOX-β-TCP-treated sites (P ≤ 0.05).EDTA root surface etching enhances DOX availability in the GCF following its release from β-TCP as a drug carrier.