Important components of reproductive efficiency are, in dairy industry, fertility and early pregnancy diagnosis on global level. The objectives of the present study were to appraise the G7G-Ovsynch protocol in postpartum Holstein cattle with or without Estrotect heat detector and to evaluate ELISA-based milk pregnancy-associated glycoproteins (PAG), optical density (OD), as a non-invasive method of pregnancy diagnosis (PD). A total of 178 multiparous postpartum cows were divided either in (1) G7G-Ovsynch-FTAI (n = 88) or (2) G7G-Ovsynch-AIDE (n = 90). Pregnancy was observed on d 30, PAI via ultrasonography (US), P4 and PAGs while on d 60, 90 with US. Pregnancy rate (PR) was compared by chi-squared. Milk PAGs and plasma P4 conc. were analyzed using REG procedures of SAS (P < 0.05). The PR on all 3 timelines was 49% (43/88) versus 55.60% (50/90); (P = 0.30) on d30, 44% (39/88) versus 49% (44/90); (P = 0.54) on d60 and 43% (38/88) versus 47% (42/90); (P = 0.70) on d90 PAI in G7G-Ovsynch-FTAI and G7G-Ovsynch-AIDE groups respectively. Pregnancy loss (PL) was 9% (4/43), 3% (1/38) in G7G-Ovsynch-FTAI versus 12% (6/50) and 5% (2/44) on d 60 (P = 0.70) and 90 (P = 0.66) in G7G-Ovsynch-AIDE group respectively. Overall PL on d90 PAI was 12 versus 16% in G7G-Ovsynch-FTAI and G7G-Ovsynch-AIDE groups respectively. The OD of milk PAGs (n = 16) was, mean ± SEM, 0.75 ± 0.08 on d30 PAI. Duplicate aliquot of milk samples that were stored at 4°C, resulted in slight reduction of OD (0.55 ± 0.057) after 60 d of storage period. P4 profile (ng/mL; mean ± SEM) was measured (n = 16) by RIA and was 6.0 ± 0.31 on d30, 6.68 ± 0.42 on d60 and 7.11 ± 0.39 on d90 PAI. Sensitivity (Se), Specificity (Sp), positive predictive value (PPV) and negative predictive value (NPV) of milk PAG-based test in present study was 100% in comparison with US (gold standard) on d30 PAI. Similarly, Se, Sp, PPV and NPV of plasma based P4 test in present study was 100% (92.6–100), 94% (84–99), 94% (84–98) and 100% respectively. In conclusion, use of Estrotect has resulted in increased PR, although non-significant, and milk PAG test is another viable PD method on d30 PAI, even with a lapse of 60 d storage.