Background: Cannabis oil titration consists of quantification of the acidic precursors tetrahydrocannabinolic acid (THCA) and cannabidiolic acid (CBDA) and their decarboxylated products, the active neutral cannabinoids delta-9-tetrahydrocannabinol (Δ9-THC) and cannabidiol (CBD), and is recommended to ensure galenic preparation quality through gas and liquid chromatography coupled with mass spectrometry (GC-MS; LC-MS). Analyses by LC-MS and GC-MS involving derivatization allow for detection of acidic and neutral cannabinoids; on the contrary, GC-MS without derivatization determines only neutral cannabinoids due to high temperature-decarboxylation occurring in the injection system. However, it is not clear if decarboxylation is complete. Methods: Different GC-MS methods with (BSTFA: TMCS and pyridine; incubation at 60 °C for 25 min) or without derivatization and an LC-MS method were developed for cannabinoid quantification. The total Δ9-THC and CBD yield of recovery were compared between the methods by testing laboratory samples with known concentrations of THCA and CBDA (total Δ9-THC and CBD: 175–351–702 ng/mL) and real cannabis oil samples (n = 6). Results: The total Δ9-THC and CBD yield of recovery were determined using LC-MS and GC-MS with derivatization, but not using GC-MS without derivatization (decarboxylation conversion rate of about 50–60%). No high deviation (<10%) in the total neutral cannabinoid concentrations in real cannabis oil samples was noticed, probably due to the low content of acidic forms in the original galenic preparation. Conclusions: This study raised awareness about the potential underestimation of the total Δ9-THC and CBD content in cannabis oils when quantification is performed by GC-MS without derivatization. The advice for pharmacists is to perform complete decarboxylation to convert all acidic precursors in neutral cannabinoids.