Silver nanoparticles (AgNPs) is widely used in commercial products due to its unique antimicrobial properties as well as physical properties. However, this wide use of AgNPs raises concerns about their presence in crops and potential human exposure via crop consumption. Unfortunately, restricted by some challenges, knowledge gaps regarding bioaccumulation of AgNPs in crop plants are still existed. One principal challenge in investigating the bioaccumulation of AgNPs in plants is their quantification in planta. To solve this challenge, we developed an organic solvent-based extraction method coupled with surface enhanced Raman spectroscopy (SERS) mapping technique to detect and quantify AgNPs in plants’ leaves. Compared with the traditional alkaline and enzymatic digestion method, the morphology of AgNPs extracted from spinach leaves could be greatly preserved. Also, the complete quantification process is much shorter than those current adopted methods. Another challenge is how to separate internalized AgNPs from surface attached AgNPs, which is a necessary step when studying the internalized AgNPs in crop plants. A combined method using sodium hypochlorite and ammonium hydroxide was developed to remove the surface attached AgNPs. Since all chemicals used in this developed washing protocol are generally considered as safe (GRAS), there is a potential to use this washing protocol as a postharvest washing procedure to reduce Ag contaminants in leafy vegetables. Taking advantage of these developed methods, we monitored the penetration capability of AgNPs with different sizes and surface coatings in spinach leaves after foliar exposure. We found the size of AgNPs is the main factor that affects the penetration depth, and the surface coating mainly affects the initial speed of interaction. For the spinach seedlings whose roots were exposed to AgNPs and Ag+, respectively, phenotypic images showed Ag+ ions are more toxic to spinach seedlings than AgNPs, indicating Ag+ ions are the major factor that contributes to the toxicity in this study. Also, the dissolution of AgNPs, as well as the reduction of Ag+ to AgNPs in Hoagland solution were observed. Through analyzing the surface ligands on these dissolved/newly formed AgNPs, we found most of the AgNPs were complexed by some thiol-containing molecules on the roots’ surfaces and few of them are present in the spinach seedlings. The information obtained in this study could provide useful guidance for effective and safe applications of AgNPs in crop plants.