To evaluate F-fluoro-2-deoxy-glucose (F-FDG) and F-fluorothymidine (F-FLT) as early-response biomarkers for phosphoinositide-3-kinase/Akt/mammalian-target-of-rapamycin (PI3K/Akt/mTOR) inhibition in breast cancer (BC) models. Two human epidermal growth factor receptor 2 (HER2)-positive (trastuzumab-sensitive SKBR3; trastuzumab-resistant JIMT1) and one triple-negative BC cell line (MDA-MB-231, trastuzumab, and everolimus resistant) were treated with trastuzumab (HER2 antagonist), PIK90 (PI3K inhibitor), or everolimus (mTOR inhibitor). Radiotracer uptake was measured before, 24, and 72 h after drug exposure and correlated with changes in cell number, glucose transporter 1 (GLUT1), cell cycle phase, and downstream signaling activation. In responsive cells, cell number correlated with F-FLT at 24 h and F-FDG at 72 h of drug exposure, except in JIMT1 treated with everolimus, where both radiotracers failed to detect response owing to a temporary increase in tracer uptake. This flare can be caused by reflex activation of Akt combined with a hyperactive insulin-like growth factor I receptor (IGF-1R) signaling, resulting in increased trafficking of GLUTs to the cell membrane (F-FDG) and enhanced DNA repair (F-FLT). In resistant cells, no major changes were observed, although a nonsignificant flair for both tracers was observed in JIMT1 treated with trastuzumab. F-FLT positron emission tomography (PET) detects response to PI3K-targeting therapy earlier than F-FDG PET in BC cells. However, therapy response can be underestimated after trastuzumab and everolimus owing to negative feedback loop and crosstalk between pathways.