The diagnosis of various histological subtypes of pituitary tumors is made using serum based hormone panel test. However, certain subtypes secrete more than one hormone, making the diagnosis ambiguous. Here, we performed 1H-NMR based metabolomic analysis of serum and whole-blood from luteinizing/follicle-stimulating (LH/FSH)-secreting (n = 24), prolactinomas (n = 14), and non-functional (NF) (n = 9) tumors. We found elevated levels of betahydroxybutyrate (BHB) in serum and whole-blood (WB) of prolactinomas (0.481 ± 0.211/0.329 ± 0.228 mM in serum/WB), but it was statistically significant (p ≤ 0.0033, Bonferroni correction) only in serum when compared with LH/FSH-secreting tumor patients (0.269 ± 0.139/0.167 ± 0.113 mM in serum/WB). Phenylalanine in NF tumors was found to be elevated in both serum and WB when compared with prolactinomas but it met the statistical significance criteria (p ≤ 0.0028) only in the serum. Alanine (p ≤ 0.011), tyrosine (p ≤ 0.014) and formate (p ≤ 0.011) were also elevated in NF tumors but none showed statistically significance when compared with prolactinomas. Quantification of BHB and the above amino acids in the circulation may aid in the development of blood-based in vitro diagnostic methods which can supplement the currently used serum hormone panel in the diagnosis of various subtypes of pituitary tumors.