The transversus abdominis plane (TAP) block causes desensitization of the abdominal wall and peritoneum. Of all the approaches proposed to perform it, the two-injection-point TAP showed the best results in terms of the area reached by the anesthetic solution. However, to date, no clinical data exist. The aim of this study was to evaluate the intra- and postoperative analgesic efficacy of a two-injection-point TAP block in dogs undergoing laparoscopic ovariectomy. A total of 26 animals were assigned to receive general inhalation anesthesia (control group), and 26 dogs were assigned to general inhalation anesthesia combined with TAP block (TAP group). The ultrasound-guided TAP block was carried out with a subcostal and cranial-to-ilium injection per hemiabdomen. The end-tidal concentration of isoflurane (EtISO) was recorded at different moments during the surgery. Postoperative pain was assessed at different time points during the first 24 h after surgery. The control group required significantly higher EtISO concentration during the ovarian resection and showed higher postoperative pain scores than the TAP group. Fewer dogs in the TAP group required intra- or postoperative rescue analgesia. TAP block can be implemented to improve postoperative pain management after laparoscopy, reducing the dosage of the systemic drugs used and, hence, their possible side effects.